Data acquisition control for advanced analytic instruments having pulsed optical sources

ABSTRACT

Instrument control and data acquisition in advanced analytic systems that utilize optical pulses for sample analysis are described. Clocking signals for data acquisition, data processing, communication, and/or other data handling functionalities can be derived from an on-board pulsed optical source, such as a passively mode-locked laser. The derived clocking signals can operate in combination with one or more clocking signals from a stable oscillator, so that instrument operation and data handling can tolerate interruptions in operation of the pulsed optical source.

RELATED APPLICATION

This Application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Ser. No. 62/685717, entitled “DATA ACQUISITIONCONTROL FOR ADVANCED ANALYTIC INSTRUMENTS HAVING PULSED OPTICAL SOURCES”filed Jun. 15, 2018, which is herein incorporated by reference in itsentirety.

FIELD

The present application relates to data acquisition and control inadvanced analytic instruments that include an optical source forproducing a sequence of optical pulses.

RELATED ART

In the area of instrumentation for sample analyses, pulsed opticalsources can be used to deliver optical excitation energy in the form ofa sequence of optical pulses to a plurality of sample wells that containsamples to be analyzed. For biochemical applications, the sample wellscan contain biological, chemical, and/or biochemical specimens that areto be analyzed. In some cases, the sample wells are configured asreaction chambers in which nucleic acid sequencing can be carried out.In other cases, the sample wells may contain other types of specimensunder study. The specimens, or a component with which the specimensreact, may be tagged with one or more fluorophores, for example, andemit radiation when excited by the optical pulses delivered to thesample wells. Detection of fluorescent emission from the sample wellscan provide information about the specimens.

SUMMARY

Apparatus and methods relating to instrument control and dataacquisition in advanced analytic systems that utilize optical pulses forsample analysis are described. In embodiments, clocking signals for dataacquisition, data processing, and/or other data handling functionalitiescan be derived from an on-board pulsed optical source, such as apassively mode-locked laser. The derived clocking signals can operate incombination with one or more clocking signals from a stable oscillator,so that instrument operation and data handling can tolerateinterruptions in operation of the pulsed optical source.

Some embodiments relate to an analytic instrument comprising a pulsedoptical source configured to output a sequence of optical pulses foranalysis of a sample and clock-generation circuitry configured toproduce a first clock signal derived from the sequence of optical pulsesand a second clock signal that is not derived from the sequence ofoptical pulses and use the first clock signal and second clock signal tovalidate data acquisitions for analysis of the sample.

Some embodiments relate to a method of operating an analytic instrument,the method comprising detecting a sequence of optical pulses andgenerating a first clock signal derived from the sequence of opticalpulses; providing the optical pulses for analysis of a sample;generating a second clock signal from an oscillator that is notsynchronized to the sequence of optical pulses; and providing the firstclock signal and second clock signal to a data processor for validatingdata acquisition operations during the analysis of the sample.

Some embodiments relate to an analytic instrument comprising aninterface module arranged to receive an optoelectronic chip that can bemounted and removed from a receptacle of the interface module, whereinthe optoelectronic chip is configured to hold a sample for analysis. Theinstrument can further include a pulsed optical source configured tooutput a sequence of optical pulses, a data processor arranged toreceive and process signals transmitted from the interface module, aclock-detection circuit having a detector arranged to detect opticalpulses produced by the pulse optical source and output a clockingsignal, and clock-generation circuitry arranged to receive the clockingsignal and output a first clock signal and a second clock signal,wherein the first clock signal is synchronized to the optical pulses andthe second clock signal is not synchronized to the optical pulses. Theinstrument can further include a first clock signal path providing thefirst clock signal to the interface module for timing data acquisitionoperations of the optoelectronic chip, a second clock signal pathproviding the second clock signal to the data processor, and a thirdclock signal path providing the first clock signal to the dataprocessor, wherein the data processor is configured to detectsynchronization discrepancies between the first clock signal and thesecond clock signal and compensate data-processing operations inresponse to detecting synchronization discrepancies.

Some embodiments relate to a method for timing charge-accumulationintervals in a photodetector. The method can comprise acts of providingoptical excitation pulses to excite a sample; generating a first clocksignal that is synchronized to the optical excitation pulses;initiating, with the first clock signal, a starting time of a firstcharge-accumulation interval for the photodetector; delaying the firstclock signal while detecting an output from the photodetector; recordingsignal levels from a first charge-accumulation interval as a function ofdelay of the first clock signal; identifying a reference point in therecorded signal levels; and setting a delay of the first clock signalsuch that the starting time is delayed from the reference point by apredetermined amount.

The foregoing and other aspects, implementations, acts, functionalities,features and, embodiments of the present teachings can be more fullyunderstood from the following description in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the figures, described herein,are for illustration purposes only. It is to be understood that in someinstances various aspects of the invention may be shown exaggerated orenlarged to facilitate an understanding of the invention. In thedrawings, like reference characters generally refer to like features,functionally similar and/or structurally similar elements throughout thevarious figures. The drawings are not necessarily to scale, emphasisinstead being placed upon illustrating the principles of the teachings.The drawings are not intended to limit the scope of the presentteachings in any way.

FIG. 1-1A is a block diagram depiction of an analytical instrument thatincludes a compact mode-locked laser module, according to someembodiments.

FIG. 1-1B depicts a compact mode-locked laser module incorporated intoan analytical instrument, according to some embodiments.

FIG. 1-2 depicts a train of optical pulses, according to someembodiments.

FIG. 1-3 depicts an example of parallel reaction chambers that can beexcited optically by a pulsed laser via one or more waveguides andcorresponding detectors for each chamber, according to some embodiments.

FIG. 1-4 illustrates optical excitation of a reaction chamber from awaveguide, according to some embodiments.

FIG. 1-5 depicts further details of an integrated reaction chamber,optical waveguide, and time-binning photodetector, according to someembodiments.

FIG. 1-6 depicts an example of a biological reaction that can occurwithin a reaction chamber, according to some embodiments.

FIG. 1-7 depicts emission probability curves for two differentfluorophores having different decay characteristics.

FIG. 1-8 depicts time-binning detection of fluorescent emission,according to some embodiments.

FIG. 1-9 depicts a time-binning photodetector, according to someembodiments.

FIG. 1-10A depicts pulsed excitation and time-binned detection offluorescent emission from a sample, according to some embodiments.

FIG. 1-10B depicts a histogram of accumulated fluorescent photon countsin various time bins after repeated pulsed excitation of a sample,according to some embodiments.

FIG. 1-11A-1-11D depict different histograms that may correspond to thefour nucleotides (T, A, C, G) or nucleotide analogs, according to someembodiments.

FIG. 2-1 depicts an example of a system for synchronizing instrumentelectronics to timing of optical pulses, according to some embodiments.

FIG. 2-2 depicts an example of clock-detection circuitry for ananalytical instrument that incorporates a pulsed optical source,according to some embodiments.

FIG. 2-3 depicts an example of clock-generation circuitry anddata-acquisition and data-processing components, according to someembodiments.

FIG. 3-1 illustrates example timing of data acquisition, according tosome embodiments.

FIG. 3-2 is an example of a normalized optical pulse profile plotted ona log scale that can be used to represent a number of photons receivedat a sample well.

FIG. 3-3 plots measured signal levels recorded for a charge-accumulationinterval of a time-binning photodetector that has been swept in timewith respect to the arrival time t_(e) of an excitation optical pulse ata sample well.

FIG. 3-4 illustrates an example of a calibration procedure for dataacquisition on an optoelectronic chip.

FIG. 4-1 depicts an example of system architecture for an advancedanalytic instrument, according to some embodiments.

FIG. 4-2 depicts instrument operation services available over a network,according to some embodiments.

The features and advantages of the present invention will become moreapparent from the detailed description set forth below when taken inconjunction with the drawings. When describing embodiments in referenceto the drawings, directional references (“above,” “below,” “top,”“bottom,” “left,” “right,” “horizontal,” “vertical,” etc.) may be used.Such references are intended merely as an aid to the reader viewing thedrawings in a normal orientation. These directional references are notintended to describe a preferred or only orientation of features of anembodied device. A device may be embodied using other orientations.

DETAILED DESCRIPTION I. Introduction

In advanced analytic systems, such as those described in U.S. patentpublication No. 2015/0141267 and in U.S. Pat. No. 9,617,594, both ofwhich are incorporated herein by reference, there can be a large numberof pixels having sample wells at which specimens are to be analyzed in amassively parallel operation. In some embodiments, the sample wells canbe integrated onto an optoelectronic chip that mounts into theinstrument. For example, the number of sample wells on such a chip canbe between about 10,000 and about 10,000,000. In some embodiments, thechip can be disposable and interchangeably mount into a receptacle of anadvanced analytic instrument by a user and interface with optical andelectronic components in the instrument. In embodiments, the instrumentcan be portable and the chip can be disposable and easily replaced by auser for each new sample analysis.

The inventors have recognized and appreciated that acquiring andhandling data collected from such a large number of sample wells in amassively parallel operation is a complex task that, if not donecorrectly, can result in failed or incorrect sample analysis. Forexample, in some applications, nucleic acid sequencing reactions may beperformed at a large number of sample wells in parallel (e.g., thousandsor millions of sample wells). During the sequencing reactions, targetnucleic acid molecules can undergo extension reactions in the samplewells and repeatedly incorporate nucleotides or nucleotide analogs intogrowing strands that are complementary to the target nucleic acidmolecules. Each incorporation event can last less than 100 milliseconds.During an incorporation event, optical emission from one or morefluorophores linked to a nucleotide can be low level and may requiremultiple excitations by optical pulses throughout the incorporationevent to obtain sufficient signal levels. Continuous detection for allactive sample wells so that incorporation events will not be missed canrequire careful timing of data acquisition on the chip, high dataacquisition rates, and large amounts of data that must be captured andtransmitted off the chip for subsequent processing.

Described herein are apparatus and methods relating to advanced analyticinstrument control and data acquisition. In embodiments, instrumentclocking signals can be derived from a pulsed optical source and usedfor aspects of data acquisition, data processing, and/or datatransmission. The described methods include steps to improve the qualityof acquired data. The apparatus and methods are useful for variousaspects of data handling in such advanced analytic instruments.

II. Example Bioanalytic Application

By way of explanation, a bioanalytic application is described in which acompact pulsed optical source (a mode-locked laser module in thisexample) is used to excite fluorophores in a plurality of reactionchambers on an optoelectronic chip. Emission from the fluorophores maybe used to determine characteristics (e.g., structure information) of aspecimen in each reaction chamber. In some cases, emission from thefluorophores may be used to determine sequence information of DNA or aprotein. The optical source and optoelectronic chip can be parts of aportable, advanced analytic instrument. In embodiments, the term“optical” may refer to ultra-violet, visible, near-infrared, andshort-wavelength infrared spectral bands. The optoelectronic chip cancarry samples to be analyzed, can be disposable, and can be easilymounted in the instrument. When mounted in a receptacle of theinstrument, the chip can be in optical and electronic communication withoptical and electronic devices within the analytic instrument. Theinstrument can also include hardware for an external interface, so thatdata from the chip can be communicated to an external network. Althoughvarious types of analyses can be performed on various samples, thefollowing explanation describes genetic sequencing. However, theinvention is not limited to instruments configured for geneticsequencing.

In overview and referring to FIG. 1-1A, a portable, advanced analyticinstrument 1-100 can comprise one or more pulsed optical sources 1-108mounted as a replaceable module within, or otherwise coupled to, theinstrument 1-100. The portable analytic instrument 1-100 can include anoptical coupling system 1-115 and an analytic system 1-160. The opticalcoupling system 1-115 can include some combination of optical components(which may include, for example, none, one, or more of each of: lens,mirror, optical filter, attenuator, beam-steering component, beamshaping component) and be configured to operate on and/or couple outputoptical pulses 1-122 from the pulsed optical source 1-108 to theanalytic system 1-160. The analytic system 1-160 can include a pluralityof components that are arranged to direct the optical pulses to at leastone sample that is to be analyzed, receive one or more optical signals(e.g., fluorescence, backscattered radiation) from the at least onesample, and produce one or more electrical signals representative of thereceived optical signals. In some embodiments, the analytic system 1-160can include one or more photodetectors and may also includesignal-processing electronics (e.g., one or more microcontrollers, oneor more field-programmable gate arrays, one or more microprocessors, oneor more digital signal processors, logic gates, etc.) configured toprocess the electrical signals from the photodetectors. The analyticsystem 1-160 can also include data transmission hardware configured totransmit and receive data to and from external devices (e.g., one ormore external devices on a network to which the instrument 1-100 canconnect via one or more data communications links). In some embodiments,the analytic system 1-160 can be configured to receive an optoelectronicchip 1-140, which holds one or more samples to be analyzed.

FIG. 1-1B depicts a further detailed example of a portable analyticalinstrument 1-100 that includes a compact pulsed optical source 1-108. Inthis example, the pulsed optical source 1-108 comprises a compact,passively mode-locked laser module 1-110. A passively mode-locked lasercan produce optical pulses autonomously, without the application of anexternal pulsed signal. In some implementations, the module can bemounted to an instrument chassis or frame 1-102, and may be locatedinside an outer casing of the instrument. According to some embodiments,a pulsed optical source 1-108 can include additional components that canbe used to operate the optical source and operate on an output beam fromthe optical source 1-108. A mode-locked laser 1-110 may comprise anelement (e.g., saturable absorber, acousto-optic modulator, Kerr lens)in a laser cavity, or coupled to the laser cavity, that induces phaselocking of the laser's longitudinal frequency modes. The laser cavitycan be defined in part by cavity end mirrors 1-111, 1-119. Such lockingof the frequency modes results in pulsed operation of the laser (e.g.,an intracavity pulse 1-120 bounces back-and-forth between the cavity endmirrors) and produces a stream of output optical pulses 1-122 from oneend mirror 1-111 which is partially transmitting.

In some cases, the analytic instrument 1-100 can be configured toreceive a removable, packaged, optoelectronic chip 1-140. The chip caninclude a plurality of reaction chambers, integrated optical componentsarranged to deliver optical excitation energy to the reaction chambers,and integrated photodetectors arranged to detect fluorescent emissionfrom the reaction chambers. In some implementations, the chip 1-140 canbe disposable, whereas in other implementations the chip can bereusable. When the chip is received by the instrument, it can be inelectrical and optical communication with the pulsed optical source andelectrical and optical communication with the analytic system 1-160.

In some embodiments, the optoelectronic chip 1-140 can be mounted (e.g.,via a socket connection) on an electronic circuit board 1-130, such as aprinted circuit board (PCB) that can include additional instrumentelectronics. For example, the PCB 1-130 can include circuitry configuredto provide electrical power, one or more clock signals, and controlsignals to the optoelectronic chip 1-140, and signal-processingcircuitry arranged to receive signals representative of fluorescentemission detected from the reaction chambers. Data returned from theoptoelectronic chip can be processed in part or entirely by electronicson the instrument 1-100, although data may be transmitted via a networkconnection to one or more remote data processors, in someimplementations. The PCB 1-130 can also include circuitry configured toreceive feedback signals from the chip relating to optical coupling andpower levels of the optical pulses 1-122 coupled into waveguides of theoptoelectronic chip 1-140. The feedback signals can be provided to oneor both of the pulsed optical source 1-108 and optical system 1-115 tocontrol one or more parameters of the output beam of optical pulses1-122. In some cases, the PCB 1-130 can provide or route power to thepulsed optical source 1-108 for operating the optical source and relatedcircuitry in the optical source 1-108.

According to some embodiments, the pulsed optical source 1-108 comprisesa compact mode-locked laser module 1-110. The mode-locked laser cancomprise a gain medium 1-105 (which can be solid-state material in someembodiments), an output coupler 1-111, and a laser-cavity end mirror1-119. The mode-locked laser's optical cavity can be bound by the outputcoupler 1-111 and end mirror 1-119. An optical axis 1-125 of the lasercavity can have one or more folds (turns) to increase the length of thelaser cavity. In some embodiments, there can be additional opticalelements (not shown in FIG. 1-1B) in the laser cavity for beam shaping,wavelength selection, and/or pulse forming. In some cases, the endmirror 1-119 comprises a saturable-absorber mirror (SAM) that inducespassive mode locking of longitudinal cavity modes and results in pulsedoperation of the mode-locked laser. The pulse repetition rate isdetermined by the length of the laser cavity (e.g., the time for anoptical pulse to make a round-trip within the laser cavity). Themode-locked laser module 1-110 can further include a pump source (e.g.,a laser diode, not shown in FIG. 1-1B) for exciting the gain medium1-105. Further details of a mode-locked laser module 1-110 can be foundin U.S. patent application Ser. No. 15/844,469, titled “CompactMode-Locked Laser Module,” filed Dec. 15, 2017, which application isincorporated herein by reference.

When the laser 1-110 is mode locked, an intracavity pulse 1-120 cancirculate between the end mirror 1-119 and the output coupler 1-111, anda portion of the intracavity pulse can be transmitted through the outputcoupler 1-111 as an output pulse 1-122. Accordingly, a train of outputpulses 1-122, as depicted in the graph of FIG. 1-2, can be detected atthe output coupler as the intracavity pulse 1-120 bounces back-and-forthbetween the output coupler 1-111 and end mirror 1-119 in the lasercavity.

FIG. 1-2 depicts temporal intensity profiles of the output pulses 1-122.In some embodiments, the peak intensity values of the emitted pulses maybe approximately equal, and the profiles may have a Gaussian temporalprofile, though other profiles such as a sech² profile may be possible.In some cases, the pulses may not have symmetric temporal profiles andmay have other temporal shapes. The duration of each pulse may becharacterized by a full-width-half-maximum (FWHM) value, as indicated inFIG. 1-2. According to some embodiments of a mode-locked laser,ultrashort optical pulses can have FWHM values less than 100 picoseconds(ps). In some cases, the FWHM values can be between approximately 5 psand approximately 30 ps.

The output pulses 1-122 can be separated by regular intervals T. Forexample, T can be determined by a round-trip travel time between theoutput coupler 1-111 and cavity end mirror 1-119. According to someembodiments, the pulse-separation interval T can be between about 1 nsand about 30 ns. In some cases, the pulse-separation interval T can bebetween about 5 ns and about 20 ns, corresponding to a laser-cavitylength (an approximate length of the optical axis 1-125 within the lasercavity) between about 0.7 meter and about 3 meters. In embodiments, thepulse-separation interval corresponds to a round trip travel time in thelaser cavity, so that a cavity length of 3 meters (round-trip distanceof 6 meters) provides a pulse-separation interval T of approximately 20ns.

According to some embodiments, a desired pulse-separation interval T andlaser-cavity length can be determined by a combination of the number ofreaction chambers on the chip 1-140, fluorescent emissioncharacteristics, and the speed of data-handling circuitry for readingdata from the optoelectronic chip 1-140. The inventors have recognizedand appreciated that different fluorophores can be distinguished bytheir different fluorescent decay rates or characteristic lifetimes.Accordingly, there needs to be a sufficient pulse-separation interval Tto collect adequate statistics for the selected fluorophores todistinguish between their different decay rates. Additionally, if thepulse-separation interval T is too short, the data handling circuitrycannot keep up with the large amount of data being collected by thelarge number of reaction chambers. The inventors have recognized andappreciated that a pulse-separation interval T between about 5 ns andabout 20 ns is suitable for fluorophores that have decay rates up toabout 2 ns and for handling data from between about 60,000 and10,000,000 reaction chambers.

According to some implementations, a beam-steering module 1-150 canreceive output pulses from the pulsed optical source 1-108 and beconfigured to adjust at least the position and incident angles of theoptical pulses onto an optical coupler of the optoelectronic chip 1-140.In some cases, the output pulses 1-122 from the pulsed optical source1-108 can be operated on by a beam-steering module 1-150 to additionallyor alternatively change a beam shape and/or beam rotation at an opticalcoupler on the optoelectronic chip 1-140. In some implementations, thebeam-steering module 1-150 can further provide focusing and/orpolarization adjustments of the beam of output pulses onto the opticalcoupler. One example of a beam-steering module is described in U.S.patent application Ser. No. 15/161,088 titled “Pulsed Laser andBioanalytic System,” filed May 20, 2016, which is incorporated herein byreference. Another example of a beam-steering module is described in aseparate U.S. patent application No. 62/435,679, filed Dec. 16, 2016 andtitled “Compact Beam Shaping and Steering Assembly,” which isincorporated herein by reference.

Referring to FIG. 1-3, the output pulses 1-122 from a pulsed opticalsource can be coupled into one or more optical waveguides 1-312 on theoptoelectronic chip. In some embodiments, the optical pulses can becoupled to one or more waveguides via one or more grating couplers1-310, though coupling to an end of one or more optical waveguides onthe optoelectronic chip can be used in some embodiments. According tosome embodiments, a quad detector 1-320 can be located on asemiconductor substrate 1-305 (e.g., a silicon substrate) for aiding inalignment of the beam of optical pulses 1-122 to a grating coupler1-310. The one or more waveguides 1-312 and sample wells or reactionchambers 1-330 can be integrated on the same semiconductor substratewith intervening dielectric layers (e.g., silicon dioxide layers)between the substrate, waveguide, reaction chambers, and photodetectors1-322.

A simplified illustration is shown in FIG. 1-3 in which a gratingcoupler 1-310 is arranged to couple incident pulses 1-122 of excitationradiation into a single waveguide 1-312. In an actual implementation,the grating coupler 1-310 can be more complex than the simplifiedstructure shown and may span several waveguides, as described in U.S.patent application Ser. No. 15/842,720 filed on Dec. 14, 2017 and titled“Compact Beam Shaping and Steering Assembly” (e.g., as described inconnection with FIG. 2-1A and FIG. 2-1B therein), which application isincorporated by reference herein in its entirety. In someimplementations, portions of the grating coupler may be offset (e.g., inthe x-direction in FIG. 1-3 herein or y-direction in FIG. 2-1A of thereferenced application) with respect to other portions of the gratingcoupler to aid in maintaining alignment of the optical beam of pulses1-122 on the grating coupler. Additionally or alternatively, portions ofthe grating coupler may have different grating periodicities withrespect to other portions of the grating coupler to aid in maintainingalignment of the optical beam of pulses 1-122 on the grating coupler.Grating couplers with offset portions and/or portions having differentgrating periodicities are described further in a U.S. provisional patentapplication No. 62/861,832 co-filed with this application by the sameApplicant on the same day, and titled “Sliced Grating Coupler withIncreased Beam Alignment Sensitivity,” which application is incorporatedby reference herein in its entirety.

Each waveguide 1-312 can include a tapered portion 1-315 below thereaction chambers 1-330 to equalize optical power coupled to thereaction chambers along the waveguide. The reducing taper can force moreoptical energy outside the waveguide's core, increasing coupling to thereaction chambers and compensating for optical losses along thewaveguide, including losses for light coupling into the reactionchambers. A second grating coupler 1-317 can be located at an end ofeach waveguide to direct optical energy to an integrated photodiode1-324. The integrated photodiode can detect an amount of power coupleddown a waveguide and provide a detected signal to feedback circuitrythat controls the beam-steering module 1-150, for example.

The sample wells 1-330 or reaction chambers 1-330 can be aligned withthe tapered portion 1-315 of the waveguide and recessed in a tub 1-340.There can be time-binning photodetectors 1-322 located on thesemiconductor substrate 1-305 for each reaction chamber 1-330. A metalcoating and/or multilayer coating 1-350 can be formed around thereaction chambers and above the waveguide to prevent optical excitationof fluorophores that are not in the reaction chambers (e.g., dispersedin a solution above the reaction chambers). The metal coating and/ormultilayer coating 1-350 may be raised beyond edges of the tub 1-340 toreduce absorptive losses of the optical energy in the waveguide 1-312 atthe input and output ends of each waveguide.

There can be a plurality of rows of waveguides, reaction chambers, andtime-binning photodetectors on the optoelectronic chip 1-140. Forexample, there can be 128 rows, each having 512 reaction chambers, for atotal of 65,536 reaction chambers in some implementations. Otherimplementations may include fewer or more reaction chambers, and mayinclude other layout configurations. Optical power from the pulsedoptical source 1-108 can be distributed to the multiple waveguides viaone or more star couplers or multi-mode interference couplers, or by anyother means, located between an optical coupler 1-310 to the chip 1-140and the plurality of waveguides 1-312.

FIG. 1-4 illustrates optical energy coupling from an optical pulse 1-122within a tapered portion of waveguide 1-315 to a reaction chamber 1-330.The drawing has been produced from an electromagnetic field simulationof the optical wave that accounts for waveguide dimensions, reactionchamber dimensions, the different materials' optical properties, and thedistance of the tapered portion of waveguide 1-315 from the reactionchamber 1-330. The waveguide can be formed from silicon nitride in asurrounding medium 1-410 of silicon dioxide, for example. The waveguide,surrounding medium, and reaction chamber can be formed bymicrofabrication processes described in U.S. application Ser. No.14/821,688, filed Aug. 7, 2015, titled “Integrated Device for Probing,Detecting and Analyzing Molecules.” According to some embodiments, anevanescent optical field 1-420 couples optical energy transported by thewaveguide to the reaction chamber 1-330.

A non-limiting example of a biological reaction taking place in areaction chamber 1-330 is depicted in FIG. 1-5. The example depictssequential incorporation of nucleotides or nucleotide analogs into agrowing strand that is complementary to a target nucleic acid. Thesequential incorporation can take place in a reaction chamber 1-330, andcan be detected by an advanced analytic instrument to sequence DNA. Thereaction chamber can have a depth between about 150 nm and about 250 nmand a diameter between about 80 nm and about 160 nm. A metallizationlayer 1-540 (e.g., a metallization for an electrical referencepotential) can be patterned above a photodetector 1-322 to provide anaperture that blocks stray light from adjacent reaction chambers andother unwanted light sources. According to some embodiments, polymerase1-520 can be located within the reaction chamber 1-330 (e.g., attachedto a base of the chamber). The polymerase can take up a target nucleicacid 1-510 (e.g., a portion of nucleic acid derived from DNA), andsequence a growing strand of complementary nucleic acid to produce agrowing strand of DNA 1-512. Nucleotides or nucleotide analogs labeledwith different fluorophores can be dispersed in a solution above andwithin the reaction chamber.

When a labeled nucleotide or nucleotide analog 1-610 is incorporatedinto a growing strand of complementary nucleic acid, as depicted in FIG.1-6, one or more attached fluorophores 1-630 can be repeatedly excitedby pulses of optical energy coupled into the reaction chamber 1-330 fromthe waveguide 1-315. In some embodiments, the fluorophore orfluorophores 1-630 can be attached to one or more nucleotides ornucleotide analogs 1-610 with any suitable linker 1-620. Anincorporation event may last for a period of time up to about 100 ms.During this time, pulses of fluorescent emission resulting fromexcitation of the fluorophore(s) by pulses from the mode-locked lasercan be detected with a time-binning photodetector 1-322, for example. Insome embodiments, there can be one or more additional integratedelectronic devices 1-323 at each pixel for signal handling (e.g.,amplification, read-out, routing, signal preprocessing, etc.). Accordingto some embodiments, each pixel can include a single or multilayeroptical filter 1-530 that passes fluorescent emission and reducestransmission of radiation from the excitation pulse. Someimplementations may not use the optical filter 1-530. By attachingfluorophores with different emission characteristics (e.g., fluorescentdecay rates, intensity, fluorescent wavelength) to the differentnucleotides (A,C,G,T), detecting and distinguishing the differentemission characteristics while the strand of DNA 1-512 incorporates anucleic acid and enables determination of the genetic sequence of thegrowing strand of DNA.

According to some embodiments, an advanced analytic instrument 1-100that is configured to analyze samples based on fluorescent emissioncharacteristics can detect differences in fluorescent lifetimes and/orintensities between different fluorescent molecules, and/or differencesbetween lifetimes and/or intensities of the same fluorescent moleculesin different environments. By way of explanation, FIG. 1-7 plots twodifferent fluorescent emission probability curves (A and B), which canbe representative of fluorescent emission from two different fluorescentmolecules, for example. With reference to curve A (dashed line), afterbeing excited by a short or ultrashort optical pulse, a probabilityp_(A)(t) of a fluorescent emission from a first molecule may decay withtime, as depicted. In some cases, the decrease in the probability of aphoton being emitted over time can be represented by an exponentialdecay function p_(A)(t)=P_(Ao)e^(−t/τ) ¹ , where P_(Ao) is an initialemission probability and τ₁ is a temporal parameter associated with thefirst fluorescent molecule that characterizes the emission decayprobability. τ₁ may be referred to as the “fluorescence lifetime,”“emission lifetime,” or “lifetime” of the first fluorescent molecule. Insome cases, the value of τ₁ can be altered by a local environment of thefluorescent molecule. Other fluorescent molecules can have differentemission characteristics than that shown in curve A. For example,another fluorescent molecule can have a decay profile that differs froma single exponential decay, and its lifetime can be characterized by ahalf-life value or some other metric.

A second fluorescent molecule may have a decay profile p_(B)(t) that isexponential, but has a measurably different lifetime τ₂, as depicted forcurve B in FIG. 1-7. In the example shown, the lifetime for the secondfluorescent molecule of curve B is shorter than the lifetime for curveA, and the probability of emission p_(B)(t) is higher sooner afterexcitation of the second molecule than for curve A. Differentfluorescent molecules can have lifetimes or half-life values rangingfrom about 0.1 ns to about 20 ns, in some embodiments.

The inventors have recognized and appreciated that differences influorescent emission lifetimes can be used to discern between thepresence or absence of different fluorescent molecules and/or to discernbetween different environments or conditions to which a fluorescentmolecule is subjected. In some cases, discerning fluorescent moleculesbased on lifetime (rather than emission wavelength, for example) cansimplify aspects of an analytical instrument 1-100. As an example,wavelength-discriminating optics (such as wavelength filters, dedicateddetectors for each wavelength, dedicated pulsed optical sources atdifferent wavelengths, and/or diffractive optics) can be reduced innumber or eliminated when discerning fluorescent molecules based onlifetime. In some cases, a single pulsed optical source operating at asingle characteristic wavelength can be used to excite differentfluorescent molecules that emit within a same wavelength region of theoptical spectrum but have measurably different lifetimes. An analyticsystem that uses a single pulsed optical source, rather than multiplesources operating at different wavelengths, to excite and discerndifferent fluorescent molecules emitting in a same wavelength region canbe less complex to operate and maintain, more compact, and can bemanufactured at lower cost.

Although analytic systems based on fluorescent lifetime analysis canhave certain benefits, the amount of information obtained by an analyticsystem and/or detection accuracy can be increased by allowing foradditional detection techniques. For example, some analytic systems1-160 can additionally be configured to discern one or more propertiesof a sample based on fluorescent wavelength and/or fluorescentintensity.

Referring again to FIG. 1-7, according to some embodiments, differentfluorescent lifetimes can be distinguished with a photodetector that isconfigured to time-bin fluorescent emission events following excitationof a fluorescent molecule. The time binning can occur during a singlecharge-accumulation cycle for the photodetector. A charge-accumulationcycle is an interval between read-out events during whichphoto-generated carriers are accumulated in bins of the time-binningphotodetector. The concept of determining fluorescent lifetime bytime-binning of emission events is introduced graphically in FIG. 1-8.At time t_(e) just prior to ti, a fluorescent molecule or ensemble offluorescent molecules of a same type (e.g., the type corresponding tocurve B of FIG. 1-7) is (are) excited by a short or ultrashort opticalpulse. For a large ensemble of molecules, the intensity of emission canhave a time profile similar to curve B, as depicted in FIG. 1-8.

For a single molecule or a small number of molecules, however, theemission of fluorescent photons occurs according to the statistics ofcurve B in FIG. 1-7, for this example. A time-binning photodetector1-322 can accumulate carriers generated from emission events intodiscrete time bins. Three bins are indicated in FIG. 1-8, though fewerbins or more bins may be used in embodiments. The bins are temporallyresolved with respect to the excitation time t_(c) of the fluorescentmolecule(s). For example, a first bin can accumulate carriers producedduring an interval between times t₁ and t₂, occurring after theexcitation event at time t_(e). A second bin can accumulate carriersproduced during an interval between times t₂ and t₃, and a third bin canaccumulate carriers produced during an interval between times t₃ and t₄.When a large number of emission events are summed, carriers accumulatedin the time bins can approximate the decaying intensity curve shown inFIG. 1-8, and the binned signals can be used to distinguish betweendifferent fluorescent molecules or different environments in which afluorescent molecule is located.

Examples of a time-binning photodetector 1-322 are described in U.S.patent application Ser. No. 14/821,656, filed Aug. 7, 2015, titled“Integrated Device for Temporal Binning of Received Photons” and in U.S.patent application Ser. No. 15/852,571, filed Dec. 22, 2017, titled“Integrated Photodetector with Direct Binning Pixel,” which are bothincorporated herein by reference in their entirety. For explanationpurposes, a non-limiting embodiment of a time-binning photodetector isdepicted in FIG. 1-9. A single time-binning photodetector 1-322 cancomprise a photon-absorption/carrier-generation region 1-902, acarrier-discharge channel 1-906, and a plurality of carrier-storage bins1-908 a, 1-908 b all formed on a semiconductor substrate.Carrier-transport channels 1-907 can connect between thephoton-absorption/carrier-generation region 1-902 and carrier-storagebins 1-908 a, 1-908 b. In the illustrated example, two carrier-storagebins are shown, but there may be more or fewer. There can be a read-outchannel 1-910 connected to the carrier-storage bins. Thephoton-absorption/carrier-generation region 1-902, carrier-dischargechannel 1-906, carrier-storage bins 1-908 a, 1-908 b, and read-outchannel 1-910 can be formed by doping the semiconductor locally and/orforming adjacent insulating regions to provide photodetectioncapability, confinement, and transport of carriers. A time-binningphotodetector 1-322 can also include a plurality of electrodes 1-920,1-921, 1-922, 1-923, 1-924 formed on the substrate that are configuredto generate electric fields in the device for transporting carriersthrough the device.

In operation, a portion of an excitation pulse 1-122 from a pulsedoptical source 1-108 (e.g., a mode-locked laser) is delivered to asample well 1-330 over the time-binning photodetector 1-322. Initially,some excitation radiation photons 1-901 may arrive at thephoton-absorption/carrier-generation region 1-902 and produce carriers(shown as light-shaded circles). There can also be some fluorescentemission photons 1-903 that arrive with the excitation radiation photons1-901 and produce corresponding carriers (shown as dark-shaded circles).Initially, the number of carriers produced by the excitation radiationcan too large compared to the number of carriers produced by thefluorescent emission. The initial carriers produced during a timeinterval |t_(e)-t₁| can be rejected by gating them into acarrier-discharge channel 1-906 with a first electrode 1-920, forexample.

At a later times mostly fluorescent emission photons 1-903 arrive at thephoton-absorption/carrier-generation region 1-902 and produce carriers(indicated a dark-shaded circles) that provide useful and detectablesignal that is representative of fluorescent emission from the samplewell 1-330. According to some detection methods, a second electrode1-921 and third electrode 1-923 can be gated at a later time to directcarriers produced at a later time (e.g., during a second time interval|t₁-t₂|) to a first carrier-storage bin 1-908 a. Subsequently, a fourthelectrode 1-922 and fifth electrode 1-924 can be gated at a later time(e.g., during a third time interval |t₂-t₃|) to direct carriers to asecond carrier-storage bin 1-908 b. Charge accumulation can continue inthis manner after excitation pulses for a large number of excitationpulses to accumulate an appreciable number of carriers and signal levelin each carrier-storage bin 1-908 a, 1-908 b. At a later time, thesignal can be read out from the bins. In some implementations, the timeintervals corresponding to each storage bin are at the sub-nanosecondtime scale, though longer time scales can be used in some embodiments(e.g., in embodiments where fluorophores have longer decay times).

The process of generating and time-binning carriers after an excitationevent (e.g., excitation pulse from a pulsed optical source) can occuronce after a single excitation pulse or be repeated multiple times aftermultiple excitation pulses during a single charge-accumulation cycle forthe time-binning photodetector 1-322. After charge accumulation iscomplete, carriers can be read out of the storage bins via the read-outchannel 1-910. For example, an appropriate biasing sequence can beapplied to electrodes 1-923, 1-924 and at least to electrode 1-940 toremove carriers from the storage bins 1-908 a, 1-908 b. The chargeaccumulation and read-out processes can occur in a massively paralleloperation on the optoelectronic chip 1-140 resulting in frames of data.

Although the described example in connection with FIG. 1-9 includesmultiple charge storage bins 1-908 a, 1-908 b in some cases a singlecharge storage bin may be used instead. For example, only binl may bepresent in a time-binning photodetector 1-322. In such a case, a singlestorage bins 1-908 a can be operated in a variable time-gated manner tolook at different time intervals after different excitation events. Forexample, after pulses in a first series of excitation pulses, electrodesfor the storage bin 1-908 a can be gated to collect carriers generatedduring a first time interval (e.g., during the second time interval|t₁-t₂|), and the accumulated signal can be read out after a firstpredetermined number of pulses. After pulses in a subsequent series ofexcitation pulses at the same sample well, the same electrodes for thestorage bin 1-908 a can be gated to collect carriers generated during adifferent interval (e.g., during the third time interval |t₂-t₃|), andthe accumulated signal can be read out after a second predeterminednumber of pulses. Carriers could be collected during later timeintervals in a similar manner if needed. In this manner, signal levelscorresponding to fluorescent emission during different time periodsafter arrival of an excitation pulse at a sample well can be producedusing a single carrier-storage bin.

Regardless of how charge accumulation is carried out for different timeintervals after excitation, signals that are read out can provide ahistogram having bins that are representative of the fluorescentemission decay characteristics, for example. An example process isillustrated in FIG. 1-10A and FIG. 1-10B. The histogram's bins canindicate a number of photons detected during each time interval afterexcitation of the fluorophore(s) in a sample well 1-330. In someembodiments, signals for the bins will be accumulated following a largenumber of excitation pulses, as depicted in FIG. 1-10A. The excitationpulses can occur at times t_(e1), t_(e2), t_(e3), . . . t_(eN) which areseparated by the pulse interval time T. In some cases, there can bebetween 10 ⁵ and 10 ⁷ excitation pulses 1-122 (or portions thereof)applied to a sample well during an accumulation of signals in theelectron-storage bins for a single event being observed in the samplewell (e.g., a single nucleotide incorporation event in DNA analysis). Insome embodiments, one bin (bin 0) can be configured to detect anamplitude of excitation energy delivered with each optical pulse, andmay be used as a reference signal (e.g., to normalize data). In othercases, the excitation pulse amplitude may be stable, determined one ormore times during signal acquisition, and not determined after eachexcitation pulse so that there is no bin0 signal acquisition after eachexcitation pulse. In such cases, carriers produced by an excitationpulse can be rejected and dumped from thephoton-absorption/carrier-generation region 1-902 as described above inconnection with FIG. 1-9.

In some implementations, only a single photon may be emitted from afluorophore following an excitation event, as depicted in FIG. 1-10A.After a first excitation event at time t_(e1), the emitted photon attime t_(f1) may occur within a first time interval (e.g. , between timest₁ and t₂), so that the resulting electron signal is accumulated in thefirst electron-storage bin (contributes to bin 1). In a subsequentexcitation event at time t_(e2), the emitted photon at time t_(f2) mayoccur within a second time interval (e.g. , between times t₂ and t₃), sothat the resulting electron signal contributes to bin 2. After a nextexcitation event at time t_(e3), a photon may emit at a time te3occurring within the first time interval.

In some implementations, there may not be a fluorescent photon emittedand/or detected after each excitation pulse received at a sample well1-330. In some cases, there can be as few as one fluorescent photon thatis detected at a sample well for every 10,000 excitation pulsesdelivered to the sample well. One advantage of implementing amode-locked laser 1-110 as the pulsed excitation source 1-108 is that amode-locked laser can produce short optical pulses having high intensityand quick turn-off times at high pulse-repetition rates (e.g., between50 MHz and 250 MHz). With such high pulse-repetition rates, the numberof excitation pulses within a 10 millisecond charge-accumulationinterval can be 50,000 to 250,000, so that detectable signal can beaccumulated.

After a large number of excitation events and carrier accumulations, thecarrier-storage bins of the time-binning photodetector 1-322 can be readout to provide a multi-valued signal (e.g., a histogram of two or morevalues, an N-dimensional vector, etc.) for a sample well. The signalvalues for each bin can depend upon the decay rate of the fluorophore.For example and referring again to FIG. 1-8, a fluorophore having adecay curve B will have a higher ratio of signal in bin 1 to bin 2 thana fluorophore having a decay curve A. The values from the bins can beanalyzed and compared against calibration values, and/or each other, todetermine the particular fluorophore present. For a sequencingapplication, identifying the fluorophore can determine the nucleotide ornucleotide analog that is being incorporated into a growing strand ofDNA, for example. For other applications, identifying the fluorophorecan determine an identity of a molecule or specimen of interest, whichmay be linked to the fluorophore.

To further aid in understanding the signal analysis, the accumulated,multi-bin values can be plotted as a histogram, as depicted in FIG.1-10B for example, or can be recorded as a vector or location inN-dimensional space. Calibration runs can be performed separately toacquire calibration values for the multi-valued signals (e.g.,calibration histograms) for four different fluorophores linked to thefour nucleotides or nucleotide analogs. As an example, the calibrationhistograms may appear as depicted in FIG. 1-11A (fluorescent labelassociated with the T nucleotide), FIG. 1-11B (fluorescent labelassociated with the A nucleotide), FIG. 1-11C (fluorescent labelassociated with the C nucleotide), and FIG. 1-11D (fluorescent labelassociated with the G nucleotide). A comparison of the measuredmulti-valued signal (corresponding to the histogram of FIG. 1-10B) tothe calibration multi-valued signals can determine the identity “T”(FIG. 1-11A) of the nucleotide or nucleotide analog being incorporatedinto the growing strand of DNA.

In some implementations, fluorescent intensity can be used additionallyor alternatively to distinguish between different fluorophores. Forexample, some fluorophores may emit at significantly differentintensities or have a significant difference in their probabilities ofexcitation (e.g., at least a difference of about 35%) even though theirdecay rates may be similar. By referencing binned signals (bins 1-3) tomeasured excitation energy and/or other acquired signals, it can bepossible to distinguish different fluorophores based on intensitylevels.

In some embodiments, different numbers of fluorophores of the same typecan be linked to different nucleotides or nucleotide analogs, so thatthe nucleotides can be identified based on fluorophore intensity. Forexample, two fluorophores can be linked to a first nucleotide (e.g.,“C”) or nucleotide analog and four or more fluorophores can be linked toa second nucleotide (e.g., “T”) or nucleotide analog. Because of thedifferent numbers of fluorophores, there may be different excitation andfluorophore emission probabilities associated with the differentnucleotides. For example, there may be more emission events for the “T”nucleotide or nucleotide analog during a signal accumulation interval,so that the apparent intensity of the bins is significantly higher thanfor the “C” nucleotide or nucleotide analog.

The inventors have recognized and appreciated that distinguishingnucleotides or any other biological or chemical specimens based onfluorophore decay rates and/or fluorophore intensities enables asimplification of the optical excitation and detection systems in ananalytical instrument 1-100. For example, optical excitation can beperformed with a single-wavelength source (e.g., a source producing onecharacteristic wavelength rather than multiple sources or a sourceoperating at multiple different characteristic wavelengths).Additionally, wavelength discriminating optics and filters may not beneeded in the detection system to distinguish between fluorophores ofdifferent wavelengths. Also, a single photodetector can be used for eachreaction chamber to detect emission from different fluorophores.

The phrase “characteristic wavelength” or “wavelength” is used to referto a central or predominant wavelength within a limited bandwidth ofradiation (e.g., a central or peak wavelength within a 20 nm bandwidthoutput by a pulsed optical source). In some cases, “characteristicwavelength” or “wavelength” may be used to refer to a peak wavelengthwithin a total bandwidth of radiation output by a source.

The inventors have recognized and appreciated that fluorophores havingemission wavelengths in a range between about 560 nm and about 900 nmcan provide adequate amounts of fluorescence to be detected by atime-binning photodetector (which can be fabricated on a silicon waferusing CMOS processes). These fluorophores can be linked to biologicalmolecules of interest, such as nucleotides or nucleotide analogs forgenetic sequencing applications. Fluorescent emission in this wavelengthrange can be detected with higher responsivity in a silicon-basedphotodetector than fluorescence at longer wavelengths. Additionally,fluorophores and associated linkers in this wavelength range may notinterfere with incorporation of the nucleotides or nucleotide analogsinto growing strands of DNA. The inventors have also recognized andappreciated that fluorophores having emission wavelengths in a rangebetween about 560 nm and about 660 nm can be optically excited with asingle-wavelength source. An example fluorophore in this range is AlexaFluor 647, available from Thermo Fisher Scientific Inc. of Waltham,Mass. The inventors have also recognized and appreciated that excitationenergy at shorter wavelengths (e.g., between about 500 nm and about 650nm) may be required to excite fluorophores that emit at wavelengthsbetween about 560 nm and about 900 nm. In some embodiments, thetime-binning photodetectors can efficiently detect longer-wavelengthemission from the samples, e.g., by incorporating other materials, suchas Ge, into the photodetectors active region.

Although the prospect of sequencing DNA using an excitation source thatemits a single characteristic wavelength can simplify some of theoptical system, it can place technically challenging demands on theexcitation source and data acquisition. For example, the inventors haverecognized and appreciated that optical pulses from the excitationsource should extinguish quickly for the detection schemes describedabove, so that the excitation energy does not overwhelm or interferewith the subsequently detected fluorescent signals that may be used todistinguish fluorophores based on lifetime and/or intensity. Theinventors have recognized and appreciated that mode-locked lasers canprovide such rapid turn-off characteristics. However, mode-locked laserscan be difficult to operate in a stable mode-locking state for extendedperiods of time, which can adversely affect signal acquisition. Forexample, when distinguishing fluorophores based on time and/orintensity, stability of data acquisition timing and excitation pulseintensity are important to reduce misrecognition errors. Followingextensive efforts, a compact and stable mode-locked laser was conceivedand implemented as a replaceable module for an advanced analyticinstrument 1-100. An example mode-locked laser module is described inU.S. patent application Ser. No. 15/844,469 referenced above. Such alaser has been found to provide a stable output of pulse intensity whenoperated continuously for hours. However, even with such a stablemode-locked laser, drift in the pulse repetition rate can occur as wellas an occasional pulse drop-out (e.g., a missing pulse).

The importance of data acquisition timing can be understood withreference again to FIG. 1-10A. In order to properly accumulatefluorescent signals in bins, it is important that the timing intervalsof the bins occur at a same time following each successive opticalexcitation pulse. If the timing of the bins drift with respect to theoptical pulse, then carriers produced in the photodetector 1-322 byfluorescent signals may be accumulated into an incorrect bin andcontribute to misrecognition of a fluorophore and a correspondingspecimen. Additionally, it is desirable to read data from theoptoelectronic chip 1-140 in approximate synchronicity with dataacquisition at the reaction chambers to avoid data overruns and misseddata.

One approach to controlling data acquisition timing is illustrated inFIG. 2-1. The inventors have recognized and appreciated that it isdesirable to synchronize at least some electronic operations (e.g., dataacquisition, signal processing, data transmission) of an analytic system1-160 with the repetition rate of optical pulses 1-122 that are outputfrom a mode-locked laser 1-110, for example. According to someembodiments, a timer 2-120 can be configured to detect optical pulses1-120 or 1-122 produced by the mode-locked laser 1-110 and produce atiming signal (e.g., a clock signal) that can be synchronized with thesequence of optical pulses 1-122 and used to trigger instrumentelectronic operations. The inventors have recognized and appreciatedthat there are at least two important aspects associated with deriving atiming signal from a mode-locked laser or other pulsed optical source. Afirst aspect is to configure the instrument electronics so that, whenusing such a timing signal, the instrument will stably and continuouslyoperate even though there may be intermittent interruptions in theproduction of optical pulses or drift in the frequency or repetitionrate of the optical pulses. A second aspect is to configure theinstrument electronics to time the occurrence of data acquisition binson the optoelectronic chip (e.g., bin1, bin2, bin3, etc.) to collecthigh-quality signals that improve the performance of the instrument indistinguishing between different fluorophores.

In FIG. 2-1, optical pulses 1-122 are depicted as being separatedspatially by a distance D. The illustration represents a snapshot intime. This separation distance corresponds to the time T between pulsesaccording to the relation T=D/c where c is the speed of light. Inpractice, the time T between pulses can be measured with a photodiodeand oscilloscope. For a mode-locked laser 1-110, the time T correspondsto a round-trip time of an optical pulse in the laser's cavity.According to some embodiments, f_(sync)=1/(TN) where f_(sync) representsthe frequency of a clock signal generated from the detected train ofoptical pulses 1-122 and N is an integer greater than or equal to 1. Insome implementations, a clock signal can be generated such thatf_(sync)=N/T where N is an integer greater than or equal to 1.

According to some embodiments, the timer 2-120 can receive an analog ordigitized signal from a photodiode that detects optical pulses 1-122from the mode-locked laser 1-110. The photodiode can be mounted in ornear the mode-locked laser 1-110 or at a location in the analyticinstrument 1-100 where it can detect light (scattered or transmitted)from the optical pulses 1-120 or 1-122. The timer 2-120 can use anysuitable method to form or trigger a synchronizing signal from thereceived analog or digitized signal. For example, the timer can use aSchmitt trigger or comparator to form a train of digital pulses fromdetected optical pulses. In some implementations, the timer 2-120 canfurther use a delay-locked loop or phase-locked loop to synchronize astable clock signal from a stable electronic clock source to a train ofdigital pulses produced from the detected optical pulses. The train ofdigital pulses and/or the locked stable clock signal can be provided tothe analytic system 1-160 to synchronize electronics on the instrument1-100 with the optical pulses.

In some embodiments, clock-detection circuitry is used to generate aclocking signal that can be used to drive data-acquisition electronicsin a portable analytic instrument 1-100. An example of a clock-detectioncircuit 2-200 is depicted in FIG. 2-2, though the invention is notlimited to the particular circuitry in the drawing. In some cases, theclock-detection circuit 2-200, or a portion thereof, can be assembled ona printed circuit board (PCB) that is part of a pulsed optical source1-108, such as a mode-locked laser module 1-110. According to someembodiments, clock-detection circuit 2-200 can include stages of pulsedetection, signal amplification with automatic gain control, clockdigitization, and clock frequency/phase locking.

A pulse-detection stage 2-205 can comprise a high-speed photodiode 2-210that is reversed biased and connected between a biasing potential and areference potential (e.g., a ground potential), according to someembodiments. The photodiode 2-210 can be connected in series with tworesistors R₁, R₂ to provide a desired amount of reverse bias, accordingto some implementations. A reverse bias on the photodiode can be anysuitable value, and can be fixed using fixed-value resistors R₁, R₂ orcan be adjustable. In some cases, a capacitor C can be connected betweena cathode of the photodiode 2-210 and a reference potential to enhancethe speed of the photodiode 2-210 and/or reduce signal noise. A signalfrom the anode of the photodiode can be provided to an amplificationstage 2-207. In some embodiments, the pulse-detection stage 2-205 can beconfigured to detect optical pulses having an average power levelbetween about 100 microwatts and about 25 milliwatts. Thepulse-detection stage 2-205 of the clock-detection circuit 2-200 can bemounted on or near the mode-locked laser 1-110, and arranged to detectoptical pulses 1-120 or 1-122 produced by the optical source (e.g.,mode-locked laser 1-110).

An amplification stage 2-207 can comprise one or more analog amplifiers2-220 that can include variable gain adjustments or adjustableattenuation, so that pulse output levels from the analog gain amplifierscan be set within a predetermined range. An amplification stage 2-207 ofthe clock-detection circuit 2-200 can further include an automatic gaincontrol amplifier 2-240. In some cases, analog filtering circuitry 2-230can be connected to an output of the analog amplifiers 2-220 (e.g., toremove high-frequency (e.g., greater than about 500 MHz) and/orlow-frequency noise (e.g., less than about 100 Hz)). The filtered orunfiltered output from the one or more analog gain amplifiers 2-220 canbe provided to an automatic gain control amplifier 2-240, according tosome embodiments.

In some cases, a final output signal from the one or more analogamplifiers can be positive-going. The inventors have recognized andappreciated that a subsequent automatic gain-control (AGC) amplifier2-240 operates more reliably when its input pulses peak to positivevoltage rather than negative voltage. The automatic gain controlamplifier 2-240 can vary its internal gain to compensate for amplitudefluctuations in the received electronic pulse train. The output pulsetrain from the automatic gain control amplifier 2-240 can haveapproximately constant amplitude, as depicted in FIG. 2-2, whereas theinput to the automatic gain control amplifier 2-240 can havefluctuations in the pulse-to-pulse amplitudes. An example automatic gaincontrol amplifier 2-240 is model AD8368 available from Analog Devices,Inc. of Norwood, Mass.

In a clock digitization stage 2-209, an output from the automatic gaincontrol amplifier 2-240 can be provided to a comparator 2-250 to producea digital pulse train 2-252, according to some implementations. Forexample, the pulse train from the AGC amplifier can be provided to afirst input of a comparator 2-250, and a reference potential (which canbe user-settable in some embodiments) can be connected to a second inputof the comparator 2-250. The reference potential can establish thetrigger point for the rising edge of each produced digital pulse.

As may be appreciated, fluctuations in received optical pulse amplitudeswould lead to fluctuations in amplitudes of the electronic pulses beforethe AGC amplifier 2-240. Without the AGC amplifier, these amplitudefluctuations would lead to timing jitter in the rising edges of pulsesin the digitized pulse train from the comparator 2-250. By leveling thepulse amplitudes with the AGC amplifier 2-240, pulse jitter after thecomparator 2-250 is reduced significantly. For example, timing jittercan be reduced to less than about 50 picoseconds with the AGC amplifier.In some implementations, an output from the comparator can be providedto logic circuitry 2-270 which is configured to change the duty cycle ofthe digitized pulse train to approximately 50%.

In some implementations, a frequency/phase-locking stage 2-211 of theclock-detection circuit 2-200 can comprise a phase-locked loop (PLL)that is used to produce at least one stable output clock signal CLK fortiming and synchronizing instrument operations to the optical pulses1-122. According to some embodiments, an output from the clockdigitization stage 2-209 can be provided to a first input of afrequency/phase detector 2-280, and a signal from a stable electronic orelectro-mechanical voltage controlled oscillator (VCO) 2-260 can beprovided to a second input of the detector 2-280. An electronic orelectro-mechanical oscillator can be highly stable against mechanicalperturbations and against temperature variations. The PLL can furtherinclude a loop filter 2-282 arranged to filter an output from thefrequency/phase detector 2-280 that is fed back to the VCO. Inembodiments, the loop filter 2-282 can effectively integrate thedetected difference signal from the frequency/phase detector 2-280 overa selected number of clock cycles.

According to some embodiments, a phase and frequency of the stable clocksignal from the VCO 2-260 can be locked by the PLL to a phase andfrequency of the digitized clock signal 0S1 derived from optical pulses1-122 of the pulsed optical source (e.g., mode-locked laser 1-110),which can be less stable. By using an integration period in the PLL thatspans multiple optical pulses, the electronic or electro-mechanicaloscillator 2-260 can lock to the frequency and phase of the opticalpulse train and ride through short-term instabilities (e.g., pulsejitter, pulse drop outs) of the mode-locked laser 1-110. In this manner,the frequency/phase-locking stage 2-211 can produce one or more stableoutput clock signals CLK that are derived from a stable electro orelectro-mechanical oscillator 2-260 and synchronized to the opticalpulses 1-120 or 1-122 produced by the optical source 1-108. In someimplementations, the output clock signal CLK can be provided to a clocksynthesis stage that can divide the clock signal M ways and synthesizedifferent clock signals from the M clock signals. An example circuitthat can be used to implement the frequency/phase-locking stage 2-211 isIC chip Si5338, which is available from Silicon Laboratories Inc. ofAustin, Tex.

The inventors have recognized and appreciated that, in someimplementations, there can be an interplay between the loop bandwidth ofthe AGC amplifier 2-240 and the loop bandwidth of the PLL in thefrequency/phase-locking stage 2-211. The loop bandwidth of the PLL isdetermined primarily by parameter values for the loop filter 2-282. Forexample, if the loop bandwidth of the PLL is too high, the output clocksignal CLK can respond to jitter introduced or passed by the AGCamplifier and comparator in the digitized pulse train, and introduceexcessive erratic behavior in the output clock signal CLK. The erraticbehavior can lead to clocking errors and instrument lock-up. On theother hand, if either or both of the AGC and PLL loop bandwidths are toolow, the resulting clock signals output from the PLL will not accuratelytrack the optical pulse timing leading to signal detection errors on theoptoelectronic chip 1-140 and unacceptably high misrecognitions ananalyzed samples. The inventors have found that an integration timeconstant associated with the loop bandwidth of the PLL should be betweenapproximately 30 pulses (±3 pulses) and approximately 80 pulses (±8pulses) of the optical pulse train from the mode-locked laser 1-110.Additionally, an integration time constant associated with the loopbandwidth of the AGC amplifier 2-240 should not exceed by more thanabout 20% the integration time constant for the PLL.

FIG. 2-3 depicts one example of clock-generation circuitry 2-311,data-acquisition, and data-handling circuitry for an advanced analyticinstrument 1-100 according to the present embodiments. Such examplecircuitry can include, but not be limited to, one or more clockgeneration circuits 2-381, 2-382, 2-383, one or more processors (such asa field-programmable gate array 2-320), memory 2-390, and acommunication interface 2-340. According to some embodiments, each clockgeneration circuit 2-381, 2-382, 2-383 may include a phase-locked loop(PLL). In embodiments, multiple clock signals can be generated and usedto time data acquisition, processing, and transmission of the data. Theinventors have found that using multiple clock signals for data handlingcan provide more stable operation of an analytic instrument 1-100.According to some implementations, one or more clock signals (CLK3,CLK4, CLK6, CLK7 in the illustrated example) are derived from and/orsynchronized to the pulsed optical source 1-108 or its sequence ofoptical pulses and can be used to drive data acquisition from theoptoelectronic chip 1-140. Additionally, one or more clock signals(CLK1, CLK2, CLK5 in the illustrated example) may not be derived fromthe pulsed optical source 1-108 or its sequence of optical pulses, andcan be derived from a stable oscillator 2-360 and can be used to drivedata processing, data acquisition, communications, and datatransmission. The inventors have recognized and appreciated that anadvanced analytic instrument 1-100 can operate more stably and betolerant of short term and long term disruptions in the pulsed opticalsource 1-108 when clock signals derived from a stable oscillator 2-360are used separately from clock signals derived from the pulsed opticalsource to drive data processing, acquisition, and communicationoperations. The inclusion of a stable oscillator can prevent instrumentlock-up due to erratic behavior of a clock signal derived from thepulsed optical source. For example, if a severe clocking error isdetected (such as one or more optical pulse drop-outs in a sequence ofoptical pulses), the clock signal source for data acquisition and/ordata processing can be switched to the stable oscillator to resumenormal operation of the instrument 1-100.

According to some embodiments, output from a stable oscillator 2-360 canbe divided with a 1:2 fan-out buffer 2-310 into two clocking signalsOSC1, OSC2 of a same frequency and provided to two clock generationcircuits 2-381, 2-382. In some cases, the clock generation circuits areprogrammable and each are capable of producing multiple output clocksignals, at least some of which have frequencies different than thefrequency of the received input signal OSC1, OSC2 at each clockgeneration circuit. There can also be at least one output signal of asame frequency as the received input signal from each clock generationcircuit 2-381, 2-382. The output clock signals of same and differentfrequencies can be derived, at least in part, from a received inputclock signal OSC1, OSC2. One example of a clock generation circuit 2-381is chip model Si5338, which is available from Silicon Laboratories Inc.of Austin, Tex.

In a first clock generation circuit 2-381, an internalvoltage-controlled oscillator (VCO) can be phase locked to a receivedstable oscillator signal OSC1 or to a periodic clocking signal OS1derived from a train of optical pulses 1-120 or 1-122. The oscillatorsignal OSC1 can be produced by a stable electrical or electro-mechanicaloscillator 2-360 (or any other suitable oscillator). In someembodiments, the clock generation circuit 2-381 can include circuitryfor implementing a PLL to lock the frequency and phase of the internalVCO to either the signal OSC1 from oscillator 2-360 or to the clockingsignal OS1 derived from the train of optical pulses. The circuitry forimplementing a phase-locked loop can include aphase/frequency-difference detector, loop filter, and the VCO, forexample. Selection of the input signal (e.g., OS1 or OSC1) forphase-locking can be performed via a control signal provided over an I²Ccommunication link, for example. Selection of the input signal maydepend upon stability or presence of the clocking signal OS1. Forexample, the signal OSC1 may be selected when the clocking signal OS1 isnot present or unstable in amplitude, frequency, phase, or a combinationthereof. In some implementations, the analytic instrument may beconfigured to automatically switch to and back from theoscillator-derived clock signal OSC1 when a disruption in the clockingsignal OS1 is detected, so that the data acquisitions can ride throughtemporary disruptions in the sequence of optical pulses. Thephase-locked loop can output a signal that is synchronized in frequencyand/or phase to either a periodic clocking signal OS1 derived from atrain of optical pulses 1-120 or 1-122 or to the stable oscillatorsignal OSC1.

According to some embodiments, the frequency of the signal OSC1 producedby the stable oscillator 2-360 can be significantly different from theperiodic clocking signal OS1. For example, the frequency of the signalOSC1 can be on the order of 10 MHz and the frequency of the signal OS1can be on the order of 65 MHz. In order to provide an output clocksignal (e.g., CLK3) from the first clock generation circuit 2-381 thatis essentially equal to a frequency f₁ of the clocking signal OS1derived from the pulsed optical source 1-108 when the stable oscillator2-360 is selected as an input signal source, the internal PLL andcircuitry of the first clock generation circuit 2-381 can be configuredto step the frequency up or down to a target value. In some cases, thefrequency values can be set via a communication interface, e.g., an I²Cinterface. Accordingly, regardless of selection of the input signalsource (whether OS1 or OSC1), the output clock frequencies can bemaintained at an essentially same value. A clock signal (e.g., CLK3)output from the first clock generation circuit 2-381 may be provided tothe optoelectronic chip 1-140 to time data acquisition operations atsample wells on the chip.

The inventors have recognized and appreciated that due to thecomplexities of the optoelectronic chip 1-140 and pulsed optical source1-108 (e.g., mode-locked laser), there are periods of operation of theadvanced analytic instrument 1-100 during which it can be preferable toperform operations with the optoelectronic chip 1-140 while the pulsedoptical source 1-108 is in an off state or a warm-up state. During theseperiods, the input signal to the first clock generation circuit 2-381can be provided from the stable oscillator 2-360. Subsequently, when thepulsed optical source 1-108 is operating and stable, the input signal tothe first clock generation circuit 2-381 can be switched from the stableoscillator 2-360 to the periodic clocking signal OS1 derived from atrain of optical pulses 1-120 or 1-122. In some operations, switchingbetween signal OS1 and signal OSC1 can be performed as a part ofautomated instrument operation (e.g., checking electronics on theoptoelectronic chip 1-140) prior to running an analysis of samples onthe chip 1-140.

When a switch is made between input signals to the first clockgeneration circuit 2-381, there can be a brief disruption in clocksignals output from the first clock generation circuit 2-381. Such adisruption can cause data transmission, data processing, and/orcommunication errors between the optoelectronic chip 1-140 and the FPGA2-320 or other data processor. In embodiments, a clock signal (e.g.,CLK3) derived from the first clock generation circuit 2-381 can beprovided to the FPGA 2-320 or other data processor to compare with aclock signal derived from the stable oscillator 2-360 (e.g., CLK1produced via a second clock generation circuit 2-382), so that the FPGAcan detect disruptions in the clock signal(s) that are provided to theoptoelectronic chip 1-140 and prevent errors in data transmission, dataprocessing, data acquisition, and/or communication, as explained furtherbelow. For example, a clock signal (e.g., CLK3) derived from the firstclock generation circuit 2-381 can be divided with a 1:2 fan-out buffer2-310 and one of the output clock signals provided to the FPGA 2-320.

In some cases, the clock generation circuit 2-381 can include circuitryfor outputting multiple clock signals CLK3, CLK4 that are produced fromthe PLL of the first clock generation circuit 2-381. The multiple clocksignals can have same or different frequencies. Multiple clock signalsof different frequencies can be produced by splitting an output signalfrom the PLL of the clock generation circuit 2-381 into multiple clocksignals of a same frequency and providing one or more of the multipleclock signals to one or more clock dividers, which may be fractional(e.g., non-integer) or integer dividers. Different divider values can beused for each divider to produce multiple clock signals having differentfrequencies and output from the clock generation circuit 2-381.

In some cases, each clock signal CLK3, CLK4 output from the clockgeneration circuit 2-381 can have essentially a same frequency f₁. Aclock generation circuit may include a programmable phase-adjust circuitthat allows for fine and independent adjustment of each output clock'sphase. According to some embodiments, programmability of phase, clockfrequencies, and other aspects of clock generation (e.g., clockselection, clock amplitude, PLL loop bandwidth) can be performed via anI²C communication link or other data communication link. A datacommunication link may be established using a communication interface2-340, such as a universal serial bus interface. Because there can be alarge number of sample wells distributed across the optoelectronic chip1-140, in some cases each clock signal CLK3, CLK4 can be provided to adifferent region of the chip to improve clock distribution and timinguniformity across the chip. For example, a clock signal provided to thechip 1-140 may be split into N clock signals of a same frequency andprovided to N different spatial locations on the chip 1-140, where N isan integer.

According to some implementations, a first clock signal CLK3 from thefirst clock generation circuit 2-381 can be split with a 1:2 fan-outbuffer 2-310 and provided to optoelectronic chip 1-140 and also to adata processor (e.g., field-programmable gate array (FPGA) 2-320) thatprocesses data received from the optoelectronic chip 1-140. A secondclock signal CLK4 can also be provided to the optoelectronic chip 1-140.In some implementations, the first clock signal CLK3 and/or second clocksignal CLK4 can be used to drive data acquisition from sample wells1-330 on the optoelectronic chip 1-140 and each may have essentially asame frequency f₁ as the train of optical pulses 1-122 that are incidenton the optoelectronic chip 1-140. For example, the first clock signalCLK3 and/or second clock signal CLK4 can be used to trigger the timingof charge-accumulation intervals (e.g., gating of electrodes) for thetime-binning photodetectors 1-322 on the optoelectronic chip 1-140, sothat charge-accumulation intervals can be synchronized to the arrivalfrequency and/or time of optical pulses 1-122 at the sample wells 1-330.

In some implementations, the first clock signal CLK3 and/or second clocksignal CLK4 can be provided to a clock-generation circuit 2-383 thatproduces two output clock signals CLK6, CLK7, which may be at one or twodifferent frequencies of the first and second clock signals CLK3, CLK4.In some cases, one clock signal CLK6 can be used to time read-out ofdata from the array of time-binning photodetectors 1-322 (e.g., to driverow and column pointers for read-out of rows of data). The second clocksignal CLK7 can be used to drive other functions performed by theoptoelectronic chip 1-140. As an example, the second clock signal CLK7can be used to drive charge accumulation at quad detectors 1-320 orphotodiodes 1-324 on the optoelectronic chip 1-140 (e.g., photodetectorsthat can sense alignment of the optical pulses 1-122 to receivingoptical structures on the optoelectronic chip 1-140). In embodiments,signals from quad detectors 1-320 and photodiodes 1-324 may not need tobe collected as frequently as signals from the sample wells 1-330, so alower data acquisition rate may be preferred to reduce an amount of dataproduced by the chip 1-140. In some implementations, a light levelincident on the quad detectors 1-320 and photodiodes 1-324 can bereduced and longer integration times used when a slower clock frequencyis used to drive data acquisition from these detectors and photodiodes,as compared to a higher clock frequency. Accordingly, a slower clockfrequency for the second clock signal CLK7 can reduce an amount ofoptical power consumed by the quad detectors 1-320 and photodiodes1-324, making more optical power available for excitation at the samplewells 1-330.

According to some embodiments, an analytic instrument 1-100 can use twoclock signals derived from different sources and separately provided toa processor to validate data acquisitions for sample analysis (e.g., bydetermining that data arrives at a processor at an expected time forsubsequent data processing). For example, a first clock signal CLK3 canbe additionally provided to an FPGA 2-320 or other suitabledata-processing device (e.g., microcontroller, microprocessor,digital-signal processor, etc.). The first clock signal CLK3 canindicate when new data is being transmitted to the FPGA 2-320, forexample. The first clock signal CLK3 may not drive data-processingoperations on the FPGA, and instead may be used to resolve timing orsynchronization discrepancies that can occur between data acquisition onthe optoelectronic chip 1-140 and data-processing operations on the FPGA2-320. In some implementations, the frequency of the pulsed opticalsource 1-108 can drift over time leading to drifts in the periodicclocking signal OS1 derived from a train of optical pulses 1-120 or1-122. This can lead to a drift in the frequency of the first clocksignal f₁. When f₁ drifts, data acquisition at the chip 1-140 cansometimes drift out of synchronicity with data-processing operations onthe FPGA 2-320 that may be driven by a second, different clock signal(e.g., clock signal CLK1). By providing the first clock signal CLK3 tothe FPGA 2-320, the FPGA can determine when data-processing operationsproduce valid output for received data. In some implementations,data-processing operations may be suspended until the arrival of arising or falling edge of the first clock signal CLK3, so as tosynchronize data-processing operations with data acquisitions on theoptoelectronic chip 1-140. In some cases, upon detecting a timing orsynchronization discrepancy, the FPGA 2-320 or suitable data processormay alter timing of data-processing operations in order to resynchronizethe data-processing operations with data received from theoptoelectronic chip 1-140. For example, the FPGA 2-320 may drop (e.g.,discard or overwrite) one or more lines or frame(s) of data receivedfrom the optoelectronic chip 1-140 in order to resynchronizedata-processing operations with a data stream received from the chip1-140. In some cases, data-processing operations may be paused by theFPGA 2-320 or suitable data processor to await arrival andsynchronization with incoming data from the chip 1-140.

In some embodiments, a first data stream DATA1 can be transmitted fromthe optoelectronic chip 1-140 to the FPGA 2-320 based on the first clocksignal CLK3 or a clock signal CLK6 derived from the first clock signalCLK3. For example, the first clock signal CLK3 may be used directly, orconverted to a different frequency and used, to clock transmission ofsample well data from the optoelectronic chip to the FPGA. The firstdata stream DATA1 can be derived from signals detected from the samplewells 1-330. In some cases, a second data stream DATA2 can betransmitted from the optoelectronic chip 1-140 to the FPGA 2-320 basedon the second clock signal CLK7. For example, the second data streamDATA2 can be derived from signals detected from the quad detectors 1-320and photodiodes 1-324.

A second clock-generation circuit 2-382 can produce additional clocksignals CLK1, CLK2, CLK5 that are used by the analytic system 1-100.According to some embodiments, the second clock-generation circuit 2-382can receive an input clock signal OSC2 that is produced by a stableoscillator 2-360. A phase-locked loop may or may not be used orimplemented in the second clock-generation circuit 2-382. In someembodiments, the second clock-generation circuit 2-382 is used togenerate multiple clock signals CLK1, CLK2, CLK5 of different desiredfrequencies. Since the clock signals output from the secondclock-generation circuit 2-382 are derived only from a stable oscillator2-360, these clock signals can run continuously without interruption andessentially without frequency drift (e.g., less than 200 parts permillion) in contrast to the clock signals produced by the first clockgeneration circuit 2-381 that are derived from the pulsed optical source1-108. Accordingly, clock signals output from the secondclock-generation circuit 2-382 are suitable for driving data handlingand communication operations continuously, thereby avoidingdata-processing and data-communication errors or disruptions due to thepulsed optical source that might otherwise cause the data-acquisitionand data-handling circuitry 2-300 to lock up or freeze.

In some embodiments, a first clock signal CLK1 from the secondclock-generation circuit 2-382 can be used to drive data-processingoperations in the FPGA 2-320. In some embodiments, the frequency f₂ ofthe first clock signal CLK1 can be higher than the frequency f₁ of thefirst clock signal CLK3 from the first clock-generation circuit 2-381that is synchronized to the train of optical pulses 1-122. A secondclock signal CLK2 at a same frequency f₂ (or different frequency in somecases) produced by the second clock-generation circuit 2-382 can be usedto drive a data-communications interface 2-340, according to someembodiments. The data communications interface may be a USB interfacethrough which I²C communications with the clock-generation circuits2-381, 2-382 can be exchanged. The inventors have recognized andappreciated that it can be highly preferable to use a clock essentiallyfree of interruptions to drive the USB interface so that communicationswith the clock-generation circuits 2-381, 2-382 can be maintained.

In some cases, a third clock signal CLK5 at a frequency f₃ produced bythe second clock-generation circuit 2-382 can be provided to the FPGA2-320 to drive data communications between the FPGA and one or moreexternal devices. For example, the third clock signal CLK5 may be usedto derive a data-transmission clock signal DCLK that drives transmissionof processed data to and retrieval of data from a double data rate (DDR)memory device 2-390. The frequency f₃ of the third clock signal CLK5 maybe less than, the same as, or greater than frequency f₂.

The inventors have recognized and appreciated that timing of dataacquisition (e.g., timing of charge-accumulation intervals of thetime-binning photodetectors 1-322 at the optoelectronic chip 1-140) isimportant for obtaining usable signals and improved results. Accordingto some embodiments, the initiation of charge-accumulation intervalsshould begin at a preferred time after the arrival of excitation pulsesat the sample wells 1-330. If the charge-accumulation intervals begintoo soon, the relevant signals for distinguishing fluorophores may beoverwhelmed by and lost in a background signal produced by the opticalexcitation pulse. If the charge-accumulation intervals begin too late,the relevant signals may be too weak and an amount of noise arrivingwith the signals can undesirably lead to an unacceptably high number ofmisrecognitions or signal-processing errors.

FIG. 3-1 illustrates an example of an excitation pulse 3-110 and timingrelationship to a fluorophore's emission probability curve 3-120. Uponexcitation of a fluorophore within a sample well 1-330, the peak of theemission probability curve 3-120 essentially occurs at the same timet_(e) that a peak of an excitation pulse 3-110 arrives at the samplewell. The emission probability curve 3-120 can be represented as afunction of time p_(B)(t-t_(e)) that decays with time from an initialvalue P_(Bo) as depicted in the drawing. In embodiments, it ispreferable to have the tail of the excitation pulse 3-110 extinguish atapproximately, or slightly before, the beginning time ti of a chargeaccumulation window for a time-binning photodetector 1-322. For theillustrated embodiment in FIG. 3-1, the charge accumulation windowextends from ti to t3, and only two time bins (|t₁-t₂|, |t₂-t₃|) areused to distinguish fluorophores.

FIG. 3-2 is an example plot of excitation photon dynamics. Theprobability of detecting an excitation photon during each picosecondwith a time-binning photodetector 1-322 is plotted, on a log scale, as afunction of time. The curve provides information about pulse dynamics ata sample well during excitation of a fluorophore. The plot was obtainedby measuring an optical pulse from a pulsed optical source with a fastphotodiode, converting the measurement result into photon number per 1picosecond time bins, and then normalizing the results to produce aprobability of detection curve having an area that sums approximatelyto 1. In this plot, the peak of the pulse arrives at time t_(e)≈200 ps.The leading edge of the pulse (left side) rises quickly in time, and thetail of the pulse (decays more slowly). The plot provides an example ofpulse shape, though other pulse shapes may be used. The plot can beconverted and/or scaled for a particular application.

For an example optoelectronic chip 1-140, the inventors have determinedthat there can be approximately 500 photons of scattered excitationradiation delivered per pulse to each sample well 1-330. Accordingly,the curve in FIG. 3-2 can be scaled upward such that its area isequivalent to approximately 500 to represent the number of scatteredphotons arriving at a sample well per pulse. These photons are unwanted,since they can contribute to a background signal from the photodetector1-322. Additionally, the curve in FIG. 3-2 can be scaled upward furtherby multiplying by the total number of pulses received during a frameintegration period to represent the number of photons arriving at asample well per frame of accumulated signal. The total number ofexcitation pulses received at a sample well during accumulation of asignal can be any number between 10 and 1,000,000. For purposes ofsignal detection, the curve in FIG. 3-2 can be corrected (e.g., scaledto account for changes in detector collection angle and quantumefficiency, to account for any optical filtering or attenuation of theexcitation wavelength that may be added) to represent a probability ofdetection of an excitation photon as a function of time.

As described above, there can be 1 or 0 fluorescent photon emitted foreach excitation pulse. Further, the inventors have observed that in somecases there can be as few as 1 fluorescent photon emitted and detectedfrom a sample well for 10,000 excitation pulses delivered to the samplewell. Accordingly for this example, to be able to detect the fluorescentphoton (if emitted), one should set the leading edge (time t₁) of afirst charge-accumulation interval at a point past the peak of the pulsewhere there is at most a probability of 10 ⁻⁴ (relative to the peakprobability) that a photon will be present. In such a case and for acorrected probability of detecting an excitation photon like the traceshown in FIG. 3-2, the time t₁ should be set at about or more than 300ps past the peak of the excitation pulse to provide a suitable rejectionratio of scattered photons from the excitation pulse. For otherconditions (e.g., different waveguide scattering amounts, differentpulse shape, different efficiency of fluorescent emission, etc.), thetime t₁ may be set at a different time relative to the peak of theexcitation pulse. As an example, a pulse shape may be different from thetrace shown in FIG. 3-2 for a different type of pulsed optical source(such as a mode-locked laser), and may fall off more rapidly past a peakof the pulse.

In practice, the inventors have found that there are other factors thatinfluence the location of the leading edge (time t₁) of a firstcharge-accumulation interval. FIG. 3-3 illustrates measured signalvalues (dark circles) obtained from a first charge-accumulation bin1-908 a of a time-binning photodetector 1-322. The signal values wereobtained by delivering excitation pulses 1-122 to a “dry” optoelectronicchip 1-140 having no sample and sweeping the phase of thedata-acquisition clock signals CLK3, CLK4. By sweeping the phase of thedata-acquisition clock signals, the timing of the firstcharge-accumulation interval |t₁-t₂| was swept in time with respect tothe arrival time t_(e) of the excitation pulse 1-122 at the sample well1-330. When the first charge-accumulation interval |t₁-t₂| straddled thearrival time t_(e) of the excitation pulse, the signal level was at amaximum value forming the plateau portion 3-320 of the curve.

When the leading edge of the excitation pulse was approaching thetrailing edge of the first charge-accumulation interval |t₁-t₂|, themeasured signal levels formed a rising edge 3-310 on the curve 3-300.When the charge-accumulation interval |t₁-t₂| moves past the peak andtrailing edge of the excitation pulse, the measured signal levels formeda falling edge 3-330 on the curve 3-300. A shoulder 3-340 was alsoobserved and is due to incomplete removal of carriers from thephoton-absorption/carrier-generation region 1-902 and other noisesources.

In embodiments and referring to FIG. 3-1, the time t_(e) of arrival ofthe excitation pulse at a sample well 1-330 and the initiation of thefluorophore's emission probability curve 3-120 are essentially lockedtogether in time. The occurrence of the times t₁, t₂, t₃ can be swept(e.g., swept together as a unit) back and forth in relative time byadjusting a phase of data-acquisition clock signals (e.g., CLK3, CLK4)that are provided to the optoelectronic chip 1-140 and used to drivecharge-accumulation cycles and signal read-out at the time-binningphotodetectors 1-322. In some implementations, the clock signals CLK3,CLK4 can be delayed by phase delay circuitry implemented on theoptoelectronic chip 1-140. The width of the time bins 3-131 (t₂-t₁),3-132 (t₃-t₂), can be set independently by circuitry on theoptoelectronic chip 1-140, according to some implementations. The widthof the time bins can be based on fluorophore decay characteristics, forexample. In some embodiments, the width of the time bins may bedetermined by numerical simulation based on the decay curves fordifferent fluorophores used, so that the bin widths increase theprobability of correctly distinguishing between the fluorophores. Forexample, the bin widths may be sized to provide a highest probability ofcorrectly distinguishing between the fluorophores. According to someimplementations, the duration of the second time bin 3-132 is greaterthan the duration of the first time bin 3-131.

According to some embodiments, a start time ti of a firstcharge-accumulation interval |t₁-t₂| can be set a predetermined time(e.g., t_(A1) at point A1) past a trailing edge (time t_(f)) of theplateau portion 3-320. The time t_(f) corresponds to a time at which thepeak of an excitation pulse (time t_(e)) approximately coincides with anend time t₂ of the first charge-accumulation window. In some cases, thetime till may be determined according to photon detection probability asdescribed in connection with FIG. 3-2. For example, the time t_(A1) canbe set to a value such that the probability of detecting one excitationphoton P_(1e) during all first charge-accumulation intervals |t₁-t₂| fora frame of data is less than the probability of detecting onefluorescent emission photon P_(1f) according to the following relation

P _(1e) ≥γ×P _(1f)

where γ may have a value between 1 and 10⁻² in some cases, between 10⁻²and 10⁻³ in some cases, and yet between 10⁻¹ and 10⁻⁴ in some cases.

According to some embodiments, a start time t₁ of a firstcharge-accumulation interval |t₁-t₂| can be set a predetermined time(e.g., t_(A2) at point A2) prior to a leading edge (time t₀) of theplateau portion 3-320. The time t₀ corresponds to a time at which thepeak of an excitation pulse (time t_(e)) approximately coincides with astart time t₁ of the charge-accumulation window. In some cases, the timet_(A2) can be determined according to photon detection probability asdescribed in connection with FIG. 3-2. For example, the time t_(A2) canbe set to a value such that the probability of detecting one excitationphoton P_(1e) during all second charge-accumulation intervals |t₂-t₃| orfinal charge-accumulation intervals for a frame of data is less than theprobability of detecting one fluorescent emission photon P_(1f)according to the following relation

P _(1e) ≥γ×P _(1f)

where γ has a value between 1 and 10⁻² in some cases, between 10⁻² and10⁻³ in some cases, and yet between 10⁻³ and 10⁻⁴ in some cases.

In some implementations, a noise characteristic of the time-binningphotodetector 1-322 may exhibit a minimum value at a time (point B)between arrivals of consecutive excitation pulses. Accordingly, apredetermined time t_(B) may be determined by sweeping the phase of thedata-acquisition clock signals CLK3, CLK4 and identifying a delay value(point B), or phase point of the resulting curve 3-300, at which aminimum in the amplitude of signal levels is received during the firstcharge-accumulation interval. The minimum signal level (point B) may bereferenced, for example, to a falling edge t_(f) of a plateau portion3-320 (e.g., time delay t_(B) from t_(f)) or to a leading edge of theplateau portion 3-320 (time t₀). Regardless of how the time t_(A1),t_(A2), or t_(B) is determined, the start time of a firstcharge-accumulation interval can be set for data acquisition by sweepingthe phase of the data-acquisition clock signals CLK3, CLK4, identifyingone or more reference points in the resulting curve 3-300, and thendelaying the start time of the first charge-accumulation interval from areference point by the selected time t_(A1), t_(A2), or t_(B). Examplesof reference points include an inflection point (such as inflectionpoints t₀ and t_(f) above), peaks, minimums, and fractional signallevels between a reference point and peak value (e.g., ½ the height on arising or falling edge from a minimum value).

Other methods for setting the start time t₁ of the firstcharge-accumulation interval can be used. Referring again to FIG. 3-1,in some embodiments, the start time t₁ can be set such that an amount ofexcitation radiation detected by one or more bins 3-131, 3-132 is nogreater than a first predetermined threshold value and no less than asecond predetermined value. For example, it may be beneficial to detecta target amount of excitation radiation in order to detect a sufficientamount of emission radiation from a sample well. According to someembodiments, the second predetermined threshold value may be no lessthan 70% of the first predetermined threshold value. In some cases, thefirst and second predetermined threshold values may be absolute signallevels (e.g., expressed in millivolts) determined for a chip 1-140 frommany measurements made with samples in the sample wells of identicalchips. The threshold values may be provided with chip information or maybe coded onto the chip for retrieval during an automatedchip-calibration procedure. Additionally or alternatively in some cases,the start time t₁ of the first charge-accumulation interval can be setsuch that a ratio of signal detected by the first bin to signal detectedby the second bin is greater than a predetermined threshold value.

The inventors have found that there can be slight timing variationsbetween optoelectronic chips 1-140 when chips are interchanged in theanalytic instrument 1-100. Even though the timing may be correct for afirst chip 1-140, the timing can be incorrect for a subsequent chip.Accordingly, a calibration procedure for each chip can be implemented toobtain a correct timing of the charge accumulation windows.

According to some embodiments, a calibration procedure can be executedfor each optoelectronic chip 1-140 before the chip is loaded with asample. Such a chip may be referred to as a “dry chip.” A calibrationprocedure may be executed by placing a dry chip in the advanced analyticinstrument 1-100 prior to loading the chip with a sample, and executingan automated chip-calibration procedure. During chip calibration, thestart time of the first charge-accumulation interval can be set asdescribed above.

During the chip-calibration procedure, in addition to setting timing forcharge-accumulation windows, optical coupling of excitation radiation tothe sample wells 1-330 and operation of time-binning photodetectors1-322 can be evaluated (e.g., to determine which sample wells 1-330 areviable for subsequent measurements). For example, an amount of signaldetected from each sample well due to the optical excitation pulses1-122 can be compared to a chip average level. The comparison can beused to identify sample wells that are not operating or exhibitingunacceptable performance. Sample wells with unacceptable performance ornot operating can be flagged by the FPGA 2-320 and data from thesesample wells can be excluded from final analysis results.

Example acts of a calibration procedure are depicted in the flow chartof FIG. 3-4. A method of calibration may comprise receiving (act 3-410)a dry optoelectronic chip 1-140 in a chip receptacle of an advancedanalytic instrument 1-100 and activating the chip electronics for dataacquisition. Activating the chip electronics can include providingelectrical power from the instrument to the chip 1-140 and receivingsignals from one or more sensors located on the chip 1-140. In someimplementations, activating the chip electronics may further compriseadjusting voltages on the chip. For example, reference values for one ormore analog-to-digital converters (ADCs) that receive analog signal(s)from one or more sensors 1-322 may be adjusted so that a full range ofeach ADC can be used to cover the range of an input analog signal.

In some implementations, the adjustment to an ADC may adjust a DC offsetor dark signal level of the ADC to increase a dynamic range of thephotodetector and ADC combination. For example, with no excitation lighton a pixel having a photodetector, a dark or baseline signal level fromthe photodetector is level shifted such that an expected full-lightsignal (filling the detector's charge-accumulation well) will be withina linear range of the ADC. The expected full-light signal can be based(to first order) on a TCAD simulation of the photodetector, according tosome embodiments. Next, the linearity and range of photodetector and ADCcan be checked during a chip check procedure by increasing an amount ofexcitation light to a full amount provided to the chip, and observingthat an output from the ADC does not saturate or clip. Further, ifclipping from the ADC is observed, then the baseline signal is levelshifted to remove the clipping. According to some embodiments, levelshift values are approximately consistent from chip to chip, so thatlevel shift values can be stored in memory and applied to each new chip.In some cases, adjustments of ADC offsets may be different for differentsections of a chip (e.g., different halves, different quadrants, etc.).

The method may further include delivering optical pulses (act 3-420) tosample wells on the chip, recording (act 3-425) signal levels during atleast a first charge-accumulation interval, and sweeping (act 3-430) aphase of a data-acquisition clock between each recorded signal level.The method may further include identifying (act 3-435) from the recordedsignal levels a time t₀ or corresponding phase point at which a starttime of the charge-accumulation window approximately coincides with apeak of the optical excitation pulses, and setting (act 3-440) the phaseof the data-acquisition clock such that the start time t₁ of the firstcharge-accumulation window is delayed by a predetermined amount. The actof identifying (act 3-435) a time to may comprising fitting a sigmoidfunction to at least a portion of the received signal levels andselecting a predetermined value of the fitted sigmoid function as thetime t₀, according to some embodiments. For example, a sigmoid functionmay be fit to a rising edge portion of signal levels illustrated in FIG.3-3. A calibration method may further include evaluating (act 3-445)signal levels for all sample wells and identifying (act 3-450) samplewells with low or abnormal signal levels for which data results shouldbe ignored.

An example of system architecture 4-100 for an advanced analyticinstrument 1-100 is depicted in FIG. 4-1. According to some embodiments,overall instrument control can be managed by a central command module4-110 that communicates with a plurality of other instrument modulesthrough various communication links (e.g., I²C, USB, ribbon cable,custom data link, etc.). In some cases, the command module 4-110 can beformed on a single PCB that mounts in the analytic instrument 1-100. ThePCB may plug into a backplane of the instrument, according to someembodiments. Command module 4-110 can comprise a data processor (e.g.,microcontroller, microprocessor, or programmable logic controller) thatis in communication with memory and programming instructions that adaptthe data processor to execute various instrument functionalities.Command module 4-110 can communicate directly with an instrument statusmanager 4-120, chip interface module 4-140, single board computer 4-160,optical source controller 4-112, and stepper controller 4-130. Arrows onthe data links indicate directions in which data can be transmittedbetween modules.

The chip interface module 4-140 can provide a data-handling interfacebetween the optoelectronic chip 1-140 and the command module 4-110, andcan relieve the command module of a data-handling burden due to thevolume of data produced by the chip 1-140. In some embodiments, theinterface module 4-140 comprises a data processor (e.g.,microcontroller, microprocessor, FPGA, programmable logic controller,logic circuitry, or some combination of these components) that is incommunication with memory and programming instructions that adapt thedata processor to carry out data-handling functionalities (e.g., datapreprocessing, data packaging, data transmission, etc.). Hardware forthe interface module 4-140 can be assembled on a single PCB that can beinstalled and replaced as a component in the analytic instrument 1-100.In some cases, the chip interface module 4-140 can be assembled on aboard that conforms with the nuclear instrumentation module (NIM)standard, so that the interface module 4-140 can plug into a backplaneof the advanced analytic instrument 1-100. In such or similar cases, areceptacle for the chip 1-140 can mount on a separate PCB andcommunicate with the interface module 4-140 via a multi-wire data link.In some implementations, the data processor of the interface module4-140 is in communication with a plurality of communication linesrunning between the chip 1-140 and the chip interface module 4-140.According to some embodiments, a chip interface module 4-140 can includea socket having hundreds of pins or pads 4-142 that contact to matingpads or pins on the chip 1-140 and enable high data transfer ratesbetween the chip 1-140 and interface module 4-140.

Other data may be provided from the optoelectronic chip 1-140 and/orinterface module 4-140 to the command module 4-110 in addition to thelarge amounts of data obtained from sample wells. Additional data caninclude temperature data from one or more thermal sensors mounted on thechip 1-140, and current and voltage data from a thermo-electric coolerthat is in thermal contact with the chip 1-140 when the chip is mountedin the analytic instrument 1-100. Additional data can also includeoptical power and alignment data measured on the chip 1-140 (e.g., dataobtained from monitoring photodiodes 1-324 and quad detectors 1-320depicted in FIG. 1-3).

Referring again to FIG. 4-1, single board computer 4-160 can include atleast one microprocessor, memory, and at least one communicationinterface that allows the single board computer 4-160 to communicatewith other external devices over a network 4-190 such as a local areanetwork, medium area network, wide area network, and/or the world-wideweb. Hardware for the single board computer can be assembled on a singlePCB that can be installed and replaced as a component in the analyticinstrument 1-100. The single board computer 4-160 can also includeprogramming instructions that adapt the single board computer to carryout data-handling functionalities (e.g., data processing, datapackaging, data transmission, data receiving, internet communications,etc.). In some implementations, there can be a direct data link or linksto shared memory between the chip interface module 4-140 and singleboard computer 4-160 for transmitting sample well data from the chip1-140 directly to the single board computer 4-160, bypassing the commandmodule 4-110. In some embodiments, FPGA 2-320 and/or DDR 2-390 may beintegrated on a same board with any one of the single board computer4-160, interface module 4-140, or command module 4-110. Single boardcomputer 4-160 can further communicate with a touch screen 4-180 thatcan provide a user interface for operating the analytic instrument1-100. In some embodiments, the single board computer 4-160 communicateswith command module 4-110 via a universal serial bus (USB) interface(e.g., a USB 3.0 interface and link).

An optical source controller 4-112 can comprise a data processor (e.g.,microcontroller, microprocessor, programmable logic controller, logiccircuitry, ASIC, or some combination of these components) that is incommunication with memory and programming instructions that adapt thesource controller 4-112 to execute functionalities for operating thepulsed optical source 1-108 (e.g., mode-locked laser 1-110). Hardwarefor the optical source controller board can be assembled on a single PCBthat can be installed and replaced as a component in the analyticinstrument 1-100. In some embodiments, the PCB may be attached to achassis on which the pulsed optical source is assembled, so that theoptical source controller 4-112 and pulsed optical source 1-108 can bereplaced as a single unit. In some cases, the optical source controller4-112 receives operation data from one or more sensors or devicesmounted on the pulsed optical source. For example the optical sourcecontroller 4-112 can receive data that indicates the position ofalignment optics (e.g., intracavity window(s), mirror(s)) within acavity of a mode-locked laser 1-110, data indicative of the position ofa half-wave plate that is used to control an amount of frequency-doubledpower output from a mode-locked laser 1-110, data indicative of anintensity level of a fundamental wavelength output from the mode-lockedlaser 1-110, data indicative of an intensity level of afrequency-doubled wavelength output from the mode-locked laser 1-110,and data indicative of a temperature of a component (e.g., gain medium)of the mode-locked laser 1-110 or pulsed optical source 1-108.

In some cases when the optical source 1-108 comprises a mode-lockedlaser, the optical source controller 4-112 also communicates with a pumpmodule 4-114 (e.g., via an I²C link). The pump module can comprise anelectro-optical assembly as described in U.S. patent application Ser.No. 15/844,469 referenced above. Data received from the pump module caninclude data indicative of the temperature of a pump source (e.g., ahigh power diode laser), data indicative of an amount of optical poweroutput by the pump source, and data indicative of operational parametersfor cooling elements (e.g., fan speed, voltage and/or current values fora thermo-electric cooler) that affect temperature of the pump source.

Optical source controller 4-112 may further communicate (e.g., via anI²C link) with clock-generation board 4-116. Example components that maybe included in a clock-generation board are described above inconnection with FIG. 2-2 and FIG. 2-3. In some cases, clock-generationcomponents can be assembled on a single PCB and mounted on a chassis onwhich the pulsed optical source 1-108 is assembled. In someimplementations, data received from the clock-generation board 4-116 caninclude data indicative of an intensity level of a fundamentalwavelength and data indicative of an intensity level of afrequency-doubled wavelength output from the pulsed optical source1-108. Additional data can include operational settings and parametersassociated with clock-generation circuits 2-381, 2-381, according tosome embodiments. Data received by the optical source controller 4-112can be communicated to the command module 4-110.

In embodiments, stepper controller 4-130 may communicate with steppermotors on a beam-steering unit 4-135. The beam-steering unit cancomprise movable optical components that are used to control the shape,position, and/or direction of the beam of optical pulses at one or morelocations within the analytic instrument 1-100. For example, steppermotors in the beam-steering unit 4-135 can be used to adjustorientations of one or more movable optical components in a beam pathbetween the pulsed optical source 1-108 and optoelectronic chip 1-140and thereby steer and position the optical pulses 1-122 with respect tothe chip 1-140. The steering and positioning of the pulses 1-122 can beexecuted automatically or semi-automatically after inserting anoptoelectronic chip 1-140 into the instrument, so as to improve opticalcoupling between the pulsed optical source 1-108 and optoelectronic chip1-140. As an example, an active feedback loop may be executed duringinstrument operation to maintain stable optical coupling of excitationradiation to the sample wells. During feedback loop operation, data fromthe optoelectronic chip 1-140 data may be received and analyzed bysingle-board computer 4-160 and/or command-module 4-110 to provideinstructions to stepper motor controller 4-130 to stabilize theposition, direction, and/or shape of the optical beam of pulses 1-122 onthe grating coupler 1-310. Stepper controller 4-130 can comprise atleast one data processor (e.g., microcontroller, microprocessor, orprogrammable logic controller) that is in communication with memory andprogramming instructions that adapt the stepper controller to activateone or more stepper motors. Hardware for the stepper controller 4-130can be assembled on a single PCB that can be installed and replaced as acomponent in the analytic instrument 1-100, according to someembodiments. In some cases, the stepper controller 4-130 can receivedata indicative of stepper motor positions, which can be communicated tothe command module 4-110.

According to some embodiments, an analytic instrument 1-100 may includeinstrument status indicators (e.g., lights, speakers, liquid-crystaldisplay(s), etc.) that provide operational status (e.g., power on light,chip present light, laser active indicator, fault indicators, etc.) ofthe analytic instrument 1-100. The status indicators may be controlledby an instrument status module 4-120 that is in communication with thecommand module 4-110 (e.g., via an I²C link). The instrument statusmodule 4-120 can comprise at least one data processor (e.g.,microcontroller, microprocessor, or programmable logic controller) thatis in communication with memory and programming instructions that adaptthe instrument status module to activate one or more indicators on theinstrument. Hardware for the instrument status module 4-120 can beassembled on a single PCB that can be installed and replaced as acomponent in the analytic instrument 1-100, according to someembodiments.

By communication with each module of the analytic instrument 1-100, thecommand module 4-110 can monitor data from instrument sensors andevaluate whether the analytic instrument 1-100 is operating correctlyand stably. Detected operation errors (e.g., chip over temperature,laser diode pump source over temperature, mode-locked laser operatingunstably) can automatically initiate corrective or safety actions (e.g.,suspend excitation of sample wells, increase cooling, turn pump sourcedown or off, realign intracavity optics to stabilize the laser).Additionally, logs of instrument sensor data can be recorded for eachmanufactured advanced analytic instrument 1-100. The sensor data logsmay be transmitted over a network 4-190 to a repository where theinformation can be evaluated to detect trends and predict behavior ofthe analytic instruments (e.g., data trends that predict subsequentunsatisfactory operation of an instrument and identify a cause of theimminent problem). Such instrument analytic data may be used to takeaction preemptively and avoid potential operating instabilities orshut-downs.

Subsets of system components can work together and essentiallyindependent of other subsets of system components to carry out specificinstrument functionality, according to embodiments. Referring again toFIG. 4-1, the optical source controller board 4-112, pump module 4-114,clock generation circuitry 4-116, and pulsed optical source 1-108 canoperated independently of other instrument components during a warm-upphase of the pulsed optical source 1-108, for example. During thiswarm-up phase, pulses 1-122 may be blocked or otherwise not output tothe optoelectronic chip 1-140 until the pulsed optical source hasstabilized. Stability of the pulsed optical source 1-108 can beevaluated by analyzing (by the optical source controller board 4-112 orthe command module 4-110) output from optical detectors that arearranged on or near the pulsed optical source 1-108 to detect outputpulses produced by the pulsed optical source 1-108. The detected outputpulses can be at a fundamental wavelength produced by the pulsed opticalsource 1-108 and/or at a converted, second-harmonic wavelength. Featuresanalyzed can include, but are not limited to, pulse amplitude stability,temperature stability of the pump module 4-114, stability ofsecond-harmonic pulse amplitude or second-harmonic power, stability of agenerated clock signal, and stability of pulse-repetition frequency.Once stable operation of the pulsed optical source 1-108 has beendetected, the optical source controller board 4-112 may initiate actsthat allow optical pulses 1-122 to be output to the optoelectronic chip1-140 and allow a clock signal that has been generated from the sequenceof optical pulses to be output to the optoelectronic chip 1-140 for dataacquisition.

Example network services 4-200 that may be accessed by an advancedanalytic instrument 1-100 are depicted in FIG. 4-2. Such networkservices may be accessible via network 4-190. Example network servicesinclude, but are not limited to, run-planning services 4-210, dataanalytic services 4-220, data storage services 4-230, and end-usersupport services 4-240. An end user 4-205, in some implementations, canaccess any of these services via the instrument's touch screen 4-180 orby a smart phone or personal computer 4-208 in communication with theanalytic instrument 1-100 via the network 4-190. Each of the servicescan be implemented, at least in part, as programming instructions,executable code, processor(s), data and/or data-storage hardware that isaccessible on or through one or more network servers as part of “cloud”based services.

In overview, run-planning services 4-210 can comprise user instructions,recommended instrument settings, instrument setting options, automatedcontrol options, etc. that aid a user 4-205 in planning and executinginstrument operation (e.g., a gene or protein sequencing run or othersample analysis run). As such, run-planning services 4-210 can include acombination of data (e.g., on-line instructions and recommendedsettings) and executable code. Example executable code may includescripts that can be downloaded to an analytic instrument 1-100,automatically configure the instrument for operation, and/or assist inexecution of a run.

Data analytic services 4-220 can comprise executable code that canreside and run on one or more servers. As an example, data analyticservices 4-220 can include on-line big data or machine-learningservices, such as Google Cloud and other big data services providers.Data analytic services 4-220 can be used to process data received fromthe sample wells on the optoelectronic chip 1-140.

Because large amounts of data can be generated from a sequencing run onthe optoelectronic chip 1-140, cloud-based data storage services 4-230may aid in handling the large volumes of data. Data storage services4-230 may comprise memory available on one or more server farms, in somecases. Data storage services 4-230 may store raw data and/orpreprocessed data from a chip 1-140 that will be subsequently analyzed(e.g., by data analytics services 4-220).

In some embodiments, data storage services 4-230 can store additionalinformation for each analytic instrument 1-100 accessed over thenetwork. For example, instrument settings for each sequencing run may bestored in data storage services 4-230, for future reference. In someimplementations, instrument sensor data logs, as described above, can bestored in data storage services 4-230 to track performance of one ormore instruments placed in service. Such sensor log information may beused to determine when instrument service may be needed and to upgradeor improve instrument operation.

End-user support services 4-240 can comprise general instrumentinformation and operation instructions made available for on-lineaccess. Further information can include trouble-shooting guidance forinstrument malfunctions. In some implementations, end-user supportservices 4-240 may include live chat sessions via the network or apublic switched telephone network to aid an end user 4-205 in operatingan analytic instrument 1-100. According to some embodiments, end-usersupport services 4-240 can include restricted access (indicated by thedashed line) to a user's analytic instrument 1-100 on a temporary orpermanent basis for remote operation by a certified technician and/orfor collection of sensor data logs.

Various configurations and methods relating to data acquisition controlfor advanced analytic instruments having pulsed optical sources arepossible as set forth in the following numbered lists of configurationsand methods.

(1) An analytic instrument comprising a pulsed optical source configuredto output a sequence of optical pulses for analysis of a sample; andclock-generation circuitry configured to produce a first clock signalderived from the sequence of optical pulses and a second clock signalthat is not derived from the sequence of optical pulses and provide thefirst clock signal and second clock signal to validate data acquisitionsfor analysis of the sample.

(2) The analytic instrument of configuration (1), further comprising aclock-detection circuit having a detector arranged to detect thesequence of optical pulses and output a clocking signal to theclock-generation circuitry; and a chip interface module having areceptacle arranged to receive an optoelectronic chip that can be placedin the receptacle by a user, wherein the optoelectronic chip isconfigured to hold the sample for analysis, and wherein theclock-generation circuitry outputs the first clock signal to the chipinterface module for timing first data acquisition operations of theoptoelectronic chip during operation of the analytic instrument.

(3) The analytic instrument of configuration (2), further comprising aphase-locked loop within the clock-generation circuitry that locks thefrequency and phase of a voltage controlled oscillator to a frequencyand phase of the clocking signal.

(4) The analytic instrument of configuration (2) or (3), wherein anintegration time of a loop filter within the phase-locked loopcorresponds to a time extending between approximately 30 of the opticalpulses and approximately 80 of the optical pulses.

(5) The analytic instrument of any one of configurations (2) through(4), wherein the clock-generation circuitry is further arranged tooutput a third clock signal to the chip interface module, wherein thethird clock signal oscillates at a frequency less than the first clocksignal and is used to time second data acquisition operations of theoptoelectronic chip during operation of the analytic instrument.

(6) The analytic instrument of configuration (5) or (6), wherein thefirst data acquisition operations comprise photodetection of fluorescentemissions from a plurality of sample wells on the optoelectronic chipand the second data acquisition operations comprise photodetection ofexcitation radiation delivered to the optoelectronic chip.

(7) The analytic instrument of configuration (5), wherein the seconddata acquisition operations comprise data indicative of alignment of anoptical beam to the optoelectronic chip.

(8) The analytic instrument of any one of configurations (1) through(7), further comprising an oscillator in the clock-generation circuitry,wherein the analytic instrument is configured to switch from using thefirst clock signal to using a third clock signal derived from theoscillator to time the data acquisitions when a disruption in thesequence of optical pulses occurs.

(9) The analytic instrument of configuration (8), wherein the analyticinstrument is configured to provide the second clock signal to the chipinterface module at a time when the pulsed optical source is notoperating.

(10) The analytic instrument of any one of configurations (1) through(9), wherein the second clock signal is derived from an electronic orelectro-mechanical oscillator.

(11) The analytic instrument of any one of configurations (1) through(10), further comprising a data processor located on the analyticinstrument that is arranged to receive and process sample analysis data.

(12) The analytic instrument of configuration (11), wherein the dataprocessor comprises a field-programmable gate array.

(13) The analytic instrument of configuration (11) or (12), wherein thedata processor is configured to receive the first clock signal and thesecond clock signal and determine whether to accept or reject at leastsome of the data acquisitions for subsequent data processing based uponthe received first and second clock signals.

(14) The analytic instrument of any one of configurations (11) thorugh(13), wherein the data processor is configured to use the second clocksignal for timing data-processing operations in the data processor.

(15) The analytic instrument of configuration (14), wherein the firstclock signal is also provided to the data processor and the dataprocessor is configured to validate the data acquisitions by comparingthe first clock signal and the second clock signal to determine whetherdata is received at a correct time for subsequent data processing by thedata processor.

(16) The analytic instrument of configuration (14) or (15), wherein thefirst clock signal is also provided to the data processor and the dataprocessor is arranged to detect synchronization discrepancies betweenthe first clock signal and the second clock signal and adjust timing ofdata-processing operations in response to detecting the synchronizationdiscrepancies.

(17) The analytic instrument of any one of configurations (14) through(16), further comprising an electronic or electro-mechanical oscillatoroperating at a frequency of oscillation that is less than a frequency ofthe first clock signal and the clock-generation circuitry translates thefrequency of oscillation of the electronic or electro-mechanicaloscillator to essentially a frequency of the first clock signal.

(18) The analytic instrument of any one of configurations (1) through(17), wherein the pulsed optical source comprises a passivelymode-locked laser that autonomously determines its pulse repetitionrate.

(19) The analytic instrument of any one of configurations (1) through(18), wherein the optical pulses are provided to an optoelectronic chipto excite fluorophores at one or more sample wells for sequencing DNA.

(20) The analytic instrument of any one of configurations (1) through(19), wherein the optical pulses are provided to an optoelectronic chipto excite fluorophores at one or more sample wells for sequencingproteins.

One or more of the above configurations may be used to implement stepsof one or more methods listed below.

(21) A method of operating an analytic instrument, the methodcomprising: detecting a sequence of optical pulses and generating afirst clock signal derived from the sequence of optical pulses;providing the optical pulses for analysis of a sample; generating asecond clock signal from an oscillator that is not synchronized to thesequence of optical pulses; and providing the first clock signal andsecond clock signal to a data processor for validating data acquisitionoperations during the analysis of the sample.

(22) The method of (21), further comprising: detecting the sequence ofoptical pulses with a detector and outputting a clocking signal based onthe detected sequence of optical pulses; deriving the first clock signalfrom the clocking signal for a first period of time; and providing to achip interface module the first clock signal, wherein the chip interfacemodule includes a receptacle arranged to receive an optoelectronic chipthat can be placed in the receptacle by a user and wherein theoptoelectronic chip is configured to hold the sample for the analysis ofthe sample.

(23) The method of (21) or (22), further comprising timing first dataacquisition operations of the optoelectronic chip during operation ofthe analytic instrument using the first clock signal.

(24) The method of (22) or (23), further comprising: deriving the firstclock signal from an oscillator for a second period of time; timingfirst data acquisition operations of the optoelectronic chip duringoperation of the analytic instrument using the first clock signal duringthe first period of time; and timing second data acquisition operationsof the optoelectronic chip during operation of the analytic instrumentusing the first clock signal during the second period of time.

(25) The method of (23) or (24), further comprising switching toderiving the first clock signal from the oscillator when a disruption inthe sequence of optical pulses occurs.

(26) The method of any one of (23) through (25), wherein deriving thefirst clock signal from an oscillator for a second period of timecomprises translating a frequency of oscillation of an electronic orelectro-mechanical oscillator to essentially a frequency of the sequenceof optical pulses.

(27) The method of any one of (21) through (26), further comprisingproviding data from the data acquisition operations to a network-baseddata analytic service.

(28) The method of any one of (21) through (27), further comprisingproviding data from sensors that monitor performance of the analyticinstrument to a network-based instrument support service.

(29) The method of any one of (21) through (28), further comprisingderiving the second clock signal from an electronic orelectro-mechanical oscillator.

(30) The method of any one of (21) through (29), further comprising:receiving, at the data processor, data from a chip interface module,wherein the chip interface module includes a receptacle arranged toreceive an optoelectronic chip that can be placed in the receptacle by auser and wherein the optoelectronic chip is configured to hold thesample for the analysis of the sample; and determining, by the dataprocessor, whether to accept or reject at least some of the data forsubsequent data processing based upon the received first and secondclock signals.

(31) The method of (30), wherein the data processor comprises afield-programmable gate array.

(32) The method of any one of (29) through (31), further comprising:detecting a synchronization discrepancy between the first clock signaland the second clock signal; and adjusting timing of data-processingoperations in response to detecting the synchronization discrepancy.

(33) The method of any one of (29) through (32), further comprisingtiming data-processing operations of the data processor with the secondclock signal.

(34) The method of any one of (21) through (33), further comprisingproducing the sequence of optical pulses with a passively mode-lockedlaser that autonomously determines its pulse repetition rate.

(35) The method of any one of (21) through (34), further comprising:outputting, by the clock-generation circuitry, a third clock signal to achip interface module, wherein the third clock signal oscillates at afrequency less than the first clock signal and wherein the chipinterface module includes a receptacle arranged to receive anoptoelectronic chip that can be placed in the receptacle by a user andwherein the optoelectronic chip is configured to hold the sample for theanalysis of the sample; timing first data acquisition operations of theoptoelectronic chip with the first clock signal; and timing second dataacquisition operations of the optoelectronic chip with the third clocksignal.

(36) The method of (35), wherein the first data acquisition operationscomprise photodetection of fluorescent emission from a plurality ofsample wells on the optoelectronic chip and the second data acquisitionoperations comprise photodetection of excitation radiation delivered tothe optoelectronic chip.

(37) A method for timing charge-accumulation intervals in aphotodetector, the method comprising: providing optical excitationpulses to excite a sample; generating a first clock signal that issynchronized to the optical excitation pulses; initiating, with thefirst clock signal, a starting time of a first charge-accumulationinterval for the photodetector; delaying the first clock signal whiledetecting an output from the photodetector; recording signal levels froma first charge-accumulation interval as a function of delay of the firstclock signal; identifying a reference point in the recorded signallevels; and setting a delay of the first clock signal such that thestarting time is delayed from the reference point by a predeterminedamount.

(38) The method of (37), wherein the photodetector has a secondcharge-accumulation interval that follows the first charge-accumulationinterval and is longer than the first charge-accumulation interval.

(39) The method of (37) or (38), wherein the predetermined amountlocates the first charge-accumulation interval such that a probabilityof detecting one excitation photon from the optical excitation pulsesfor all first charge-accumulation intervals for a frame of data is lessthan the probability of detecting one emission photon from the sample.

(40) The method of any one of (37) through (39), wherein thepredetermined amount locates a start time of the firstcharge-accumulation interval approximately at a minimum value of therecorded signal levels.

(41) The method of any one of (37) through (40), further comprising:delivering the optical excitation pulses to a plurality of sample wellson an optoelectronic chip; and executing the acts of initiating,delaying, recording, identifying, and setting for a plurality ofphotodetectors on the optoelectronic chip.

(42) The method of (41), further comprising determining whether a samplewell is operable based on an amount of signal from the opticalexcitation pulses detected at the sample well.

(43) The method of (41) or (42), further comprising: generating a secondclock signal having a frequency different from the first clock signal;and providing the second clock signal to the optoelectronic chip,wherein the second clock signal controls the starting times ofcharge-accumulation intervals for one or more alignment photodetectorsthat sense alignment of the optical excitation pulses to theoptoelectronic chip.

(44) The method of any one of (41) through (43), wherein the first clocksignal is derived from a clock-detection circuit that detects a sequenceof the optical excitation pulses.

(45) The method of any one of (41) through (44), further comprisingclocking transmission of data from the sample wells to a data processorusing the first clock signal.

IV. Conclusion

Having thus described several aspects of several embodiments of systemarchitecture for an advanced analytic system 1-100, it is to beappreciated that various alterations, modifications, and improvementswill readily occur to those skilled in the art. Such alterations,modifications, and improvements are intended to be part of thisdisclosure, and are intended to be within the spirit and scope of theinvention. While the present teachings have been described inconjunction with various embodiments and examples, it is not intendedthat the present teachings be limited to such embodiments or examples.On the contrary, the present teachings encompass various alternatives,modifications, and equivalents, as will be appreciated by those of skillin the art.

While various inventive embodiments have been described and illustrated,those of ordinary skill in the art will readily envision a variety ofother means and/or structures for performing the function and/orobtaining the results and/or one or more of the advantages described,and each of such variations and/or modifications is deemed to be withinthe scope of the inventive embodiments described. More generally, thoseskilled in the art will readily appreciate that all parameters,dimensions, materials, and configurations described are meant to beexamples and that the actual parameters, dimensions, materials, and/orconfigurations will depend upon the specific application or applicationsfor which the inventive teachings is/are used. Those skilled in the artwill recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific inventive embodimentsdescribed. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, inventiveembodiments may be practiced otherwise than as specifically describedand claimed. Inventive embodiments of the present disclosure may bedirected to each individual feature, system, system upgrade, and/ormethod described. In addition, any combination of two or more suchfeatures, systems, and/or methods, if such features, systems, systemupgrade, and/or methods are not mutually inconsistent, is includedwithin the inventive scope of the present disclosure.

Further, though some advantages of the present invention may beindicated, it should be appreciated that not every embodiment of theinvention will include every described advantage. Some embodiments maynot implement any features described as advantageous. Accordingly, theforegoing description and drawings are by way of example only.

All literature and similar material cited in this application,including, but not limited to, patents, patent applications, articles,books, treatises, and web pages, regardless of the format of suchliterature and similar materials, are expressly incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The section headings used are for organizational purposes only and arenot to be construed as limiting the subject matter described in any way.

Also, the technology described may be embodied as a method, of which atleast one example has been provided. The acts performed as part of themethod may be ordered in any suitable way. Accordingly, embodiments maybe constructed in which acts are performed in an order different thanillustrated, which may include performing some acts simultaneously, eventhough shown as sequential acts in illustrative embodiments.

All definitions, as defined and used, should be understood to controlover dictionary definitions, definitions in documents incorporated byreference, and/or ordinary meanings of the defined terms.

Numerical values and ranges may be described in the specification andclaims as approximate or exact values or ranges. For example, in somecases the terms “about,” “approximately,” and “substantially” may beused in reference to a value. Such references are intended to encompassthe referenced value as well as plus and minus reasonable variations ofthe value. For example, a phrase “between about 10 and about 20” isintended to mean “between exactly 10 and exactly 20” in someembodiments, as well as “between 10±δ1 and 20±δ2” in some embodiments.The amount of variation δ1, δ2 for a value may be less than 5% of thevalue in some embodiments, less than 10% of the value in someembodiments, and yet less than 20% of the value in some embodiments. Inembodiments where a large range of values is given, e.g., a rangeincluding two or more orders of magnitude, the amount of variation δ1,δ2 for a value could be as high as 50%. For example, if an operablerange extends from 2 to 200, “approximately 80” may encompass valuesbetween 40 and 120 and the range may be as large as between 1 and 300.When exact values are intended, the term “exactly” is used, e.g.,“between exactly 2 and exactly 200.” The term “essentially” is used toindicate within 3% of a target value.

The term “adjacent” may refer to two elements arranged within closeproximity to one another (e.g., within a distance that is less thanabout one-fifth of a transverse or vertical dimension of a larger of thetwo elements). In some cases there may be intervening structures orlayers between adjacent elements. In some cases adjacent elements may beimmediately adjacent to one another with no intervening structures orelements.

The indefinite articles “a” and “an,” as used in the specification andin the claims, unless clearly indicated to the contrary, should beunderstood to mean “at least one.”

The phrase “and/or,” as used in the specification and in the claims,should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used shall only be interpreted as indicating exclusive alternatives(i.e. “one or the other but not both”) when preceded by terms ofexclusivity, such as “either,” “one of,” “only one of,” or “exactly oneof.” “Consisting essentially of,” when used in the claims, shall haveits ordinary meaning as used in the field of patent law.

As used in the specification and in the claims, the phrase “at leastone,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively.

The claims should not be read as limited to the described order orelements unless stated to that effect. It should be understood thatvarious changes in form and detail may be made by one of ordinary skillin the art without departing from the spirit and scope of the appendedclaims. All embodiments that come within the spirit and scope of thefollowing claims and equivalents thereto are claimed.

1. An analytic instrument comprising: a pulsed optical source configuredto output a sequence of optical pulses for analysis of a sample; andclock-generation circuitry configured to produce a first clock signalderived from the sequence of optical pulses and a second clock signalthat is not derived from the sequence of optical pulses and provide thefirst clock signal and second clock signal to validate data acquisitionsfor analysis of the sample.
 2. The analytic instrument of claim 1,further comprising: a clock-detection circuit having a detector arrangedto detect the sequence of optical pulses and output a clocking signal tothe clock-generation circuitry; a chip interface module having areceptacle arranged to receive an optoelectronic chip that can be placedin the receptacle by a user, wherein the optoelectronic chip isconfigured to hold the sample for analysis, and wherein theclock-generation circuitry outputs the first clock signal to the chipinterface module for timing first data acquisition operations of theoptoelectronic chip during operation of the analytic instrument.
 3. Theanalytic instrument of claim 2, further comprising a phase-locked loopwithin the clock-generation circuitry that locks the frequency and phaseof a voltage controlled oscillator to a frequency and phase of theclocking signal.
 4. The analytic instrument of claim 3, wherein anintegration time of a loop filter within the phase-locked loopcorresponds to a time extending between approximately 30 of the opticalpulses and approximately 80 of the optical pulses.
 5. The analyticinstrument of claim 2, wherein the clock-generation circuitry is furtherarranged to output a third clock signal to the chip interface module,wherein the third clock signal oscillates at a frequency less than thefirst clock signal and is used to time second data acquisitionoperations of the optoelectronic chip during operation of the analyticinstrument.
 6. The analytic instrument of claim 5, wherein the firstdata acquisition operations comprise photodetection of fluorescentemissions from a plurality of sample wells on the optoelectronic chipand the second data acquisition operations comprise photodetection ofexcitation radiation delivered to the optoelectronic chip.
 7. Theanalytic instrument of claim 5, wherein the second data acquisitionoperations comprise data indicative of alignment of an optical beam tothe optoelectronic chip.
 8. The analytic instrument of claim 1, furthercomprising an oscillator in the clock-generation circuitry, wherein theanalytic instrument is configured to switch from using the first clocksignal to using a third clock signal derived from the oscillator to timethe data acquisitions when a disruption in the sequence of opticalpulses occurs.
 9. The analytic instrument of claim 8, wherein theanalytic instrument is configured to provide the second clock signal tothe chip interface module at a time when the pulsed optical source isnot operating.
 10. The analytic instrument of claim 1, wherein thesecond clock signal is derived from an electronic or electro-mechanicaloscillator.
 11. The analytic instrument of claim 1, further comprising adata processor located on the analytic instrument that is arranged toreceive and process sample analysis data.
 12. The analytic instrument ofclaim 11, wherein the data processor comprises a field-programmable gatearray.
 13. The analytic instrument of claim 11, wherein the dataprocessor is configured to receive the first clock signal and the secondclock signal and determine whether to accept or reject at least some ofthe data acquisitions for subsequent data processing based upon thereceived first and second clock signals.
 14. The analytic instrument ofclaim 11, wherein the data processor is configured to use the secondclock signal for timing data-processing operations in the dataprocessor.
 15. The analytic instrument of claim 14, wherein the firstclock signal is also provided to the data processor and the dataprocessor is configured to validate the data acquisitions by comparingthe first clock signal and the second clock signal to determine whetherdata is received at a correct time for subsequent data processing by thedata processor.
 16. The analytic instrument of claim 14, wherein thefirst clock signal is also provided to the data processor and the dataprocessor is arranged to detect synchronization discrepancies betweenthe first clock signal and the second clock signal and adjust timing ofdata-processing operations in response to detecting the synchronizationdiscrepancies.
 17. The analytic instrument of claim 14, furthercomprising an electronic or electro-mechanical oscillator operating at afrequency of oscillation that is less than a frequency of the firstclock signal and the clock-generation circuitry translates the frequencyof oscillation of the electronic or electro-mechanical oscillator toessentially a frequency of the first clock signal.
 18. The analyticinstrument of claim 1, wherein the pulsed optical source comprises apassively mode-locked laser that autonomously determines its pulserepetition rate.
 19. The analytic instrument of claim 1, wherein theoptical pulses are provided to an optoelectronic chip to excitefluorophores at one or more sample wells for sequencing DNA.
 20. Theanalytic instrument of claim 1, wherein the optical pulses are providedto an optoelectronic chip to excite fluorophores at one or more samplewells for sequencing proteins.
 21. A method of operating an analyticinstrument, the method comprising: detecting a sequence of opticalpulses and generating a first clock signal derived from the sequence ofoptical pulses; providing the optical pulses for analysis of a sample;generating a second clock signal from an oscillator that is notsynchronized to the sequence of optical pulses; and providing the firstclock signal and second clock signal to a data processor for validatingdata acquisition operations during the analysis of the sample.
 22. Themethod of claim 21, further comprising: detecting the sequence ofoptical pulses with a detector and outputting a clocking signal;deriving the first clock signal from the clocking signal for a firstperiod of time; and providing to a chip interface module the first clocksignal, wherein the chip interface module includes a receptacle arrangedto receive an optoelectronic chip that can be placed in the receptacleby a user and wherein the optoelectronic chip is configured to hold thesample for the analysis of the sample.
 23. The method of claim 22,further comprising timing first data acquisition operations of theoptoelectronic chip during operation of the analytic instrument usingthe first clock signal.
 24. The method of claim 22, further comprising:deriving the first clock signal from an oscillator for a second periodof time; timing first data acquisition operations of the optoelectronicchip during operation of the analytic instrument using the first clocksignal during the first period of time; and timing second dataacquisition operations of the optoelectronic chip during operation ofthe analytic instrument using the first clock signal during the secondperiod of time.
 25. The method of claim 24, further comprising switchingto deriving the first clock signal from the oscillator when a disruptionin the sequence of optical pulses occurs.
 26. The method of claim 24,wherein deriving the first clock signal from an oscillator for a secondperiod of time comprises translating a frequency of oscillation of anelectronic or electro-mechanical oscillator to essentially a frequencyof the sequence of optical pulses.
 27. The method of claim 22, furthercomprising providing data from the data acquisition operations to anetwork-based data analytic service.
 28. The method of claim 22, furthercomprising providing data from sensors that monitor performance of theanalytic instrument to a network-based instrument support service. 29.The method of claim 21, further comprising deriving the second clocksignal from an electronic or electro-mechanical oscillator.
 30. Themethod of claim 21, further comprising: receiving, at the dataprocessor, data from a chip interface module, wherein the chip interfacemodule includes a receptacle arranged to receive an optoelectronic chipthat can be placed in the receptacle by a user and wherein theoptoelectronic chip is configured to hold the sample for the analysis ofthe sample; and determining, by the data processor, whether to accept orreject at least some of the data for subsequent data processing basedupon the received first and second clock signals.
 31. The method ofclaim 30, wherein the data processor comprises a field-programmable gatearray.
 32. The method of claim 30, further comprising: detecting asynchronization discrepancy between the first clock signal and thesecond clock signal; and adjusting timing of data-processing operationsin response to detecting the synchronization discrepancy.
 33. The methodof claim 30, further comprising timing data-processing operations of thedata processor with the second clock signal.
 34. The method of claim 21,further comprising producing the sequence of optical pulses with apassively mode-locked laser that autonomously determines its pulserepetition rate.
 35. The method of claim 21, further comprising:outputting, by the clock-generation circuitry, a third clock signal to achip interface module, wherein the third clock signal oscillates at afrequency less than the first clock signal and wherein the chipinterface module includes a receptacle arranged to receive anoptoelectronic chip that can be placed in the receptacle by a user andwherein the optoelectronic chip is configured to hold the sample for theanalysis of the sample; timing first data acquisition operations of theoptoelectronic chip with the first clock signal; and timing second dataacquisition operations of the optoelectronic chip with the third clocksignal.
 36. The method of claim 35, wherein the first data acquisitionoperations comprise photodetection of fluorescent emission from aplurality of sample wells on the optoelectronic chip and the second dataacquisition operations comprise photodetection of excitation radiationdelivered to the optoelectronic chip.
 37. A method for timingcharge-accumulation intervals in a photodetector, the method comprising:providing optical excitation pulses to excite a sample; generating afirst clock signal that is synchronized to the optical excitationpulses; initiating, with the first clock signal, a starting time of afirst charge-accumulation interval for the photodetector; delaying thefirst clock signal while detecting an output from the photodetector;recording signal levels from a first charge-accumulation interval as afunction of delay of the first clock signal; identifying a referencepoint in the recorded signal levels; and setting a delay of the firstclock signal such that the starting time is delayed from the referencepoint by a predetermined amount.
 38. The method of claim 37, wherein thephotodetector has a second charge-accumulation interval that follows thefirst charge-accumulation interval and is longer than the firstcharge-accumulation interval.
 39. The method of claim 37, wherein thepredetermined amount locates the first charge-accumulation interval suchthat a probability of detecting one excitation photon from the opticalexcitation pulses for all first charge-accumulation intervals for aframe of data is less than the probability of detecting one emissionphoton from the sample.
 40. The method of claim 37, wherein thepredetermined amount locates a start time of the firstcharge-accumulation interval approximately at a minimum value of therecorded signal levels.
 41. The method of claim 37, further comprising:delivering the optical excitation pulses to a plurality of sample wellson an optoelectronic chip; and executing the acts of initiating,delaying, recording, identifying, and setting for a plurality ofphotodetectors on the optoelectronic chip.
 42. The method of claim 41,further comprising determining whether a sample well is operable basedon an amount of signal from the optical excitation pulses detected atthe sample well.
 43. The method of claim 41, further comprising:generating a second clock signal having a frequency different from thefirst clock signal; and providing the second clock signal to theoptoelectronic chip, wherein the second clock signal controls thestarting times of charge-accumulation intervals for one or morealignment photodetectors that sense alignment of the optical excitationpulses to the optoelectronic chip.
 44. The method of claim 41, whereinthe first clock signal is derived from a clock-detection circuit thatdetects a sequence of the optical excitation pulses.
 45. The method ofclaim 41, further comprising clocking transmission of data from thesample wells to a data processor using the first clock signal.